Low-molecular-compound for improving production, maintenance and proliferation of pluripotent stem cells, composition comprising the same, and culture method

ABSTRACT

Provided herein are novel indoleacrylic acid-based compounds, and pharmaceutically acceptable salts thereof, useful for the production, maintenance and proliferation of pluripotent stem cells. Also provided are cell culture compositions comprising these compounds, and methods of using these compounds in the production and maintenance of pluripotent stem cells.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.14/358,672, filed May 15, 2014, which is a 35 U.S.C. § 371 filing ofInternational Patent Application No. PCT/KR2012/009420, filed Nov. 8,2012, which claims priority to Korean Patent Application No.10-2012-0056881, filed May 29, 2012, and Korean Patent Application No.10-2012-0057803, filed May 30, 2012, the entire disclosures of which arehereby incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to an indoleacrylic acid-based novelcompound of formula 1, a cell culture medium comprising the same, acomposition for promoting reprogramming comprising the same, a cellculture comprising the same, and a method for producing and maintainingreprogrammed pluripotent stem cells using the same.

BACKGROUND ART

As used herein, the term “stem cell” generally refers to cells that haveexcellent self-renewal potential while maintaining an undifferentiatedstate and are capable of differentiating in a tissue-specific manner soas to have certain functions and shapes under certain environments andconditions. Human pluripotent stem cells, including human embryonic stemcells and human induced pluripotent stem cells, are capable ofself-renewal under suitable in vitro culture conditions and have apluripotent ability to differentiate into all types of cells of thebody. Due to such characteristics, the results of studies on thesepluripotent stem cells have been applied not only for the understandingof biological basic knowledge, including the development,differentiation and growth of organisms, but also for the development ofcell therapy agents for fundamental treatment of various diseases andthe development of new drugs. While efforts have been increasingly madeto develop practically applicable technology based on human pluripotentstem cells in various fields, there are still problems to be solved interms of efficiency, safety and economy in a process for the productionand proliferative culture of human pluripotent stem cells. Specifically,it is required to develop a technology for producing large amounts ofundifferentiated and differentiated stem cells, which can satisfy thedemand for the stem cells at any time. Particularly, for the developmentof cell therapy agents, it is necessary to ensure cell culturetechnology, which has excellent performance, can provide clinicallyapplicable cells and is highly efficient.

Generally, undifferentiated human pluripotent stem cells can becontinuously cultured by co-culturing with feeder cells such as mouseembryonic fibroblasts (MEFs) or in feeder-free conditions usingconditioned media (CM) obtained from cultures of MEFs or chemicallydefined media. However, co-culture with animal feeder cells or the useof conditioned media from animal feeder cells involves the risk oftransmitting one or more infectious agents such as viruses to humanpluripotent stem cells. For this reason, in recent years, continuedefforts have been made to develop chemically defined media containingknown components such as low-molecular-weight compounds, peptides or thelike without containing animal feeder cells or sera and apply thesechemically defined media for the production and culture of humanpluripotent stem cells. These chemically defined media havesignificantly contributed to the growth of the stem cell market.

Embryonic stem cells derived from the inner cell mass of frozen-thawedembryos are extracted from frozen-thawed embryos to be discarded, andthus pose no legal problems. However, because these cells are extractedfrom embryos, these cells pose ethical and religious issues from theviewpoint of life's destruction. In addition, because these cells arederived from limited embryos, transplant rejection cannot be avoided dueto the lack of immune compatibility between individuals. As analternative to overcome such problems, a method of producing inducedpluripotent stem cells having characteristics almost similar to those ofembryonic stem cells from adult stem cells using reprogramming factorswas recently successfully developed (Cell 126, 663-676, 2006; Cell 131,861-872, 2007; Nature 441, 1061-1067, 2006; Nature 451, 141-146, 2008).Due to the development of this method, the expectation of development ofpractically applicable stem cell technology based on pluripotent stemcells is increasing. Particularly, induced pluripotent stem cells do notrequire embryos for the production thereof, the use of the patient's ownextracted stem cells does not pose the problem of immune rejection, andthus induced pluripotent stem cells are technically highly useful.However, in order to allow the current technology to enter the stage ofpractical stage, it is necessarily required to develop technologycapable of replacing the use of virus in order to overcome the problemsof the current technology, including low efficiency of reprogramming andlow clinical safety.

As methods for improving the efficiency of reprogramming, examples ofsuccess in increasing the efficiency of reprogramming by the control ofextracellular environments or the use of additives such aslow-molecular-weight compounds have been reported. In addition, it wasreported that the efficiency of reprogramming of somatic cells iseffectively increased in a hypoxic condition similar to the environmentof embryonic stem cells (Cell Stem Cell, 5: 237-241, 2009). Also, Dr.Ding's team (Shi et al., Cell Stem Cell, 2008) reported thatlow-molecular-weight compounds such as BIX-01294 (G9a histonemethyltransferase inhibitor) and BayK8644 (L-type calcium channelagonist), RG108(DNA methyltransferase inhibitor) are effective inincreasing the efficiency of reprogramming, and Dr. Melton's team(Huangfu et al., Nat Biotechnol, 2008) reported thatlow-molecular-weight compounds such as VPA (histone deacetylaseinhibitor), TSA (histone deacetylase inhibitor) and SAHA (histonedeacetylase inhibitor are effective in increasing the efficiency ofreprogramming. With respect to the development of alternatives to theuse of virus, the following study results were reported: 1) transientexpression technology utilizing a single nonviral polycistronic vector(Gonzalez et al, PNAS USA, 2009; Chang et al, Stem cells, 2009); 2)adenoviral transfection technology (Stadtfeld et al, Science 2008); 3)establishment of iPSC using a single nonviral polycistronic vectorthrough the development of a Cre/loxP recombinant expression controlsystem (Soldner et al, Cell, 2009), and removal of a reprogrammingcassette by Cre transfection (Kaji et al, Nature, 2009), 4) a piggyback(PB) transposon system (Woltjen et al, Nature, 2009; Kaji et al, Nature,2009), and 5) nonintegrating episomal vectors (Yu et al, Science, 2009).However, the possibility of genetic abnormalities and tumorigenesisstill exists.

Accordingly, the present inventors have conducted extensive studies todiscover not only low-molecular-weight compounds capable of improvingthe technology for the maintenance and culture of undifferentiated humanpluripotent stem cells, but also low-molecular-weight compounds capableof improving the reprogramming technology of producing human pluripotentstem cells from somatic cells. As a result, the present inventors havediscovered the novel low-molecular-weight compound RSC-133 and haveverified that the use of a medium composition containing RSC-133significantly increases the efficiency of reprogramming for producinghuman pluripotent stem cells and significantly improves cultureconditions for maintaining and proliferating human pluripotent stemcells in an undifferentiated state, thereby completing the presentinvention.

DISCLOSURE Technical Problem

It is an object of the present invention to provide a novel compound offormula 1 or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a cell culturemedium comprising the novel compound of formula 1 or a pharmaceuticallyacceptable salt thereof.

Still another object of the present invention is to provide acomposition for promoting the reprogramming of differentiated cells intopluripotent stem cells, the composition comprising the novel compound offormula 1 or a pharmaceutically acceptable salt thereof.

Still another object of the present invention is to provide an in vitrocell culture comprising: differentiated cells; the composition forpromoting reprogramming; and a reprogramming factor, wherein thedifferentiated cells are reprogrammed into pluripotent stem cells.

Still another object of the present invention is to provide a method ofproducing reprogrammed pluripotent cells from differentiated cells, themethod comprising the steps of: (a) transferring a reprogramming factorto the differentiated cells; and (b) culturing the differentiated cellsin a medium containing the composition for promoting reprogramming.

Still another object of the present invention is to provide a cellculture medium capable of improving the maintenance and proliferation ofundifferentiated pluripotent stem cells.

Still another object of the present invention is to provide a method ofculturing pluripotent stem cells in an undifferentiated state.

Still another object of the present invention is to provide a cellculture medium capable of improving the maintenance and proliferation ofundifferentiated pluripotent stem cells.

Still another object of the present invention is to provide an in vitrocell culture comprising: pluripotent stem cells; and the cell culturemedium serving to maintain the pluripotent stem cells in anundifferentiated state by a plurality of continuous subcultures.

Technical Solution

In order to accomplish the above objects, in one aspect, the presentinvention provides a compound of the following formula 1 or apharmaceutically acceptable salt thereof:

wherein R₁ is a methoxy group (OCH₃), an amino group (NH₂), amethylamino group (NHCH₃), a dimethylamino group (N(CH₃)₂), anO-isopropyl group, a propylamino group (NHCH(CH₃)₂), a hydroxyl group(OH), NHCH₂-furan, NHCH₂CH₂-piperidine, or NH(CH₂)₃-morpholine; R₂ is ahydroxyl group (OH) or hydrogen (H); and the portion indicated by - - -may be a single or double bond.

In an embodiment, in formula 1, R₁ may be a methoxy group (OCH₃); R₂ maybe hydrogen; and the portion indicated by - - - may be a double bond.

In another embodiment, in formula 1, R₁ may be an amino group (NH₂); R₂may be hydrogen; and the portion indicated by - - - may be a doublebond.

In still another embodiment, in formula 1, R₁ may be a methylamino group(NHCH₃); R₂ may be hydrogen; and the portion indicated by - - - may be adouble bond.

In still another embodiment, in formula 1, R₁ may be a dimethylaminogroup (N(CH₃)₂); R₂ may be hydrogen; and the portion indicated by - - -may be a double bond.

In still another embodiment, in formula 1, R₁ may be an amino group(NH₂); R₂ may be hydrogen; and the portion indicated by - - - may be asingle bond.

In still another embodiment, in formula 1, R₁ may be O-isopropyl; R₂ maybe a hydroxyl group (OH); and the portion indicated by - - - may be adouble bond.

In still another embodiment, in formula 1, R₁ may be an amino group(NH₂); R₂ may be a hydroxyl group (OH); and the portion indicatedby - - - may be a double bond.

In still another embodiment, in formula 1, R₁ may be a methylamino group(NHCH₃); R₂ may be a hydroxyl group (OH); and the portion indicatedby - - - may be a double bond.

In still another embodiment, in formula 1, R₁ may be a propylamino group(NHCH(CH₃)₂); R₂ may be a hydroxyl group (OH); and the portion by - - -may be a double bond.

In still another embodiment, in formula 1, R₁ may be a dimethylaminogroup (NH(CH₃)₂); R₂ may be a hydroxyl group (OH); and the portionindicated by - - - may be a double bond.

In still another embodiment, in formula 1, R₁ may be a hydroxyl group(OH); R₂ may be hydrogen; and the portion indicated by - - - may be adouble bond.

In still another embodiment, in formula 1, R₁ may be NHCH₂-furan; R₂ maybe hydrogen; and the portion indicated by - - - may be a double bond.

In still another embodiment, in formula 1, R₁ may beNHCH₂CH₂-piperidine; R₂ may be hydrogen; and the portion indicatedby - - - may be a double bond.

In still another embodiment, in formula 1, R₁ may beNH(CH₂)₃-morpholine; R₂ may be hydrogen; and the portion indicatedby - - - may be a double bond.

In still another embodiment, in formula 1, R₁ may be O-isopropyl; R₂ maybe hydrogen; and the portion indicated by - - - may be a double bond.

In an embodiment, preferred examples of the compound of formula 1according to the present invention include the following compounds 1) to15):

-   1) methyl 3-[3-(1H-indol-3-yl)acrylamido]benzoate;-   2) 3-[3-(1H-indol-3-yl)acrylamido]benzamide;-   3) 3-[3-(1H-indol-3-yl)acrylamido]-N-methylbenzamide;-   4) 3-[3-(1H-indol-3-yl)acrylamido]-N,N-dimethylbenzamide;-   5) 3-(3-1H-indol-3-yl-propionylamino)-benzamide;-   6) isopropyl 3-[3-(1H-indol-3-yl)acrylamido]-4-hydroxybenzoate;-   7) 3-[3-(1H-indol-3-yl)acrylamido]-4-hydroxybenzamide);-   8) 3-[3-(1H-indol-3-yl)acrylamido]-4-hydroxy-N-methylbenzamide;-   9) 3-[3-(1H-indol-3-yl)acrylamido]-4-hydroxy-N-isopropylbenzamide;-   10) 3-[3-(1H-indol-3-yl)acrylamido]-4-hydroxy-N,N-dimethylbenzamide;-   11) 3-[3-(1H-indol-3-yl)acrylamido]benzoic acid;-   12) 3-[3-(1H-indol-3-yl)acrylamido]-N-(furan-2-ylmethyl)benzamide;-   13)    3-(3-1H-Indol-3-yl-acryloylamino)-N-(2-piperidin-1-yl-ethyl)-benzamide;-   14)    3-(3-1H-Indol-3-yl-acryloylamino)-N-(3-morpholin-4-yl-propyl)-benzamide;    and-   15) isopropyl 3-[3-(1H-indol-3-yl)acrylamido]benzoate.

The compounds of formula 1 according to the present invention may beprepared in the form of pharmaceutically acceptable salts or solvatesusing conventional methods known in the art.

The salt is preferably an acid addition salt formed by apharmaceutically acceptable free acid. The acid addition salt isprepared according to a conventional method. For example, it is preparedby dissolving the compound in an aqueous solution of an excess of acidand precipitating the formed salt using a water-miscible organicsolvent, for example, methanol, ethanol, acetone or acetonitrile.Alternatively, it may be prepared by heating the same molar amounts ofthe compound and acid or alcohol (e.g., glycol monomethylether) inwater, and then drying the mixture by evaporation or filtering theprecipitated salt by suction.

Herein, the free acid may be organic acid or inorganic acid. Examples ofthe inorganic acid include, but are not limited to, hydrochloric acid,phosphoric acid, sulfuric acid, nitric acid and tartaric acid. Examplesof the organic acid include, but are not limited to, methanesulfonicacid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleicacid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaricacid, mandelic acid, propionic acid, citric acid, lactic acid, glycolicacid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid,glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillicacid and hydroiodic acid.

In addition, the compounds of formula 1 according to the presentinvention may be prepared in the form of pharmaceutically acceptablemetal salts using bases. An alkali metal or alkaline earth metal saltmay be prepared, for example, by dissolving the compound in a solutionof an excess of alkali metal hydroxide or alkaline earth metalhydroxide, filtering out undissolved compound salt, and then drying thefiltrate by evaporation. For pharmaceutical purposes, the metal saltprepared is preferably sodium, potassium or calcium salt, but is notlimited thereto. In addition, a silver salt corresponding to the metalsalt can be obtained by reacting an alkali metal or alkaline earth metalsalt with a suitable silver salt (e.g., silver nitrate).

Pharmaceutically acceptable salts of the compound of formula 1 include,unless otherwise indicated, a salt of an acidic or basic group, whichcan be present in the compound of formula 1. Examples ofpharmaceutically acceptable salts include sodium, calcium and potassiumsalts of a hydroxyl group, and other pharmaceutically acceptable saltsof an amino group include hydrobromide, sulfate, hydrogen sulfate,phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate,citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) andp-toluenesulfonate (tosylate) salts. These salts can be prepared bymethods known in the art.

Preferably, the compound of the present invention may be3-[3-(1H-indol-3-yl)-acrylamido]-benzamide having a structure of thefollowing formula 2:

The compound of formula 2 that is an indole derivative may be named“3-(3-1H-Indol-3-yl)-acrylamido)-benzamide)” (hereinafter also referredto as Reprogramming Stimulating Compound-133, RSC-133, ID-133). It is anovel compound discovered during analog library screening oftrans-3-indoleacrylic acid-based compounds. In a process ofreprogramming mouse fibroblasts, the cells were treated with each of 39trans-3-indoleacrylic acid-based compounds, and two compounds having anexcellent effect of improving reprogramming efficiency were selected.Also, 32 new trans-3-indoleacrylic acid-based compounds having similarchemical structures, including the two selected compounds, were selectedand analyzed for the effect of improving the efficiency of reprogrammingof human fibroblasts, and based on the results of the analysis,reprogramming stimulating compound (RSC)-133 having an excellent effectof improving the efficiency of reprogramming was selected.

In an example of the present invention, in order to prepare the RSC-133compound, trans-3-indoleacrylic acid and 3-amino-benzamide weredissolved in DMF, and thenbenzotriazol-1-yl-N-oxy-tris(pyrrolidino)-phosphoniumhexafluorophosphate (PyBOP) and N,N-diisopropylethylamine (DIPEA) wereadded to the solution. The reaction solution was stirred overnight atroom temperature. The stirred solution was separated and purified toobtain 3-[3-(1H-indol-3-yl)-acrylamido]-benzamide (RSC-133) as a yellowsolid (Example 1-2).

In another aspect, the present invention provides a cell culture mediumcomprising the compound of formula 1 or a pharmaceutically acceptablesalt thereof. Preferably, the compound of formula 1 may be3-[3-(1H-indol-3-yl)-acrylamido]-benzamide (RSC-133) or apharmaceutically acceptable salt thereof.

As used herein, the term “cell culture medium” refers to a mediumcapable of supporting the growth and survival of stem cells under invitro culture conditions and is meant to include all conventional media,which are suitable for the culture of cells and used in the art. Thus,the term “cell culture medium” means a material that is added to aculture medium, such as the compound of formula 1 or a pharmaceuticallyacceptable salt thereof, in order to culture cells or achieve specificpurposes. The composition of the culture medium and culture conditionsmay be selected depending on the kind of cells. The culture medium thatis used for cell culture is preferably a cell culture minimum medium(CCMM) that generally contains a carbon source, a nitrogen source andtrace elements. Examples of the cell culture minimum medium include, butare not limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM(Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10,F-12, α-MEM (α-Minimal essential Medium), GMEM (Glasgow's Minimalessential Medium), and Iscove's Modified Dulbecco's Medium, etc.

In still another aspect, the present invention provides a compositionfor promoting the reprogramming of differentiated cells into pluripotentstem cells, the composition comprising the compound of formula 1 or apharmaceutically acceptable salt thereof. Preferably, the compound offormula 1 may be 3-[3-(1H-indol-3-yl)-acrylamido]-benzamide (RSC-133) ora pharmaceutically acceptable salt thereof.

As used herein, the term “differentiation” refers to a phenomenon inwhich the structure or function of cells is specialized during thedivision, proliferation and growth thereof. That is, the term refers toa process in which the feature or function of cell or tissue of anorganism changes in order to perform work given to the cell or tissue.For example, a process in which pluripotent stem cells such as embryonicstem cells change to ectoderm, mesoderm and endoderm cells is alsodefined as differentiation, and in a narrow sense, a process in whichhematopoietic stem cells change to red blood cells, white blood cells,platelets or the like also corresponds to differentiation.

As used herein, the term “differentiated cells” refers to cells thatundergone the differentiation process so as to have a specific shape andfunction. Differentiated cells that are used in the present inventionare not specifically limited, but are preferably somatic cells orprogenitor cells. In addition, differentiated cells are human cells.

As used herein, the term “somatic cells” refers to any differentiatedcells other than germ cells, which constitute animals or plants and havea chromosome number of 2n.

As used herein, the term “progenitor cells” refers to undifferentiatedprogenitor cells which do not express a differentiated differentiationphenotype when their progeny cells express a specific differentiatedphenotype. For example, progenitor cells for neurons are interneurons,and progenitor cells for myotube cells are myoblast.

As used herein, the term “pluripotent stem cells” refers to cells thatare capable of differentiating into all the tissues of the body and haveself-renewal potential and include embryonic stem cells and inducedpluripotent stem cells, but is not limited thereto. Pluripotent stemcells in the present invention include those derived from humans,monkeys, pigs, horses, cattle, sheep, dogs, cats, mice, rabbits or thelike. Preferably, pluripotent stem cells are human pluripotent stemcells.

As used herein, the term “embryonic stem cells” refers to pluripotent ortotipotent cells that are obtained by in vitro culture of inner cellmasses extracted from blastocysts immediately before implantation intothe uterus of the mother, are capable of differentiating into all thetissues of the body, and have self-renewal potential. In a broad sense,the term also includes embryoid bodies derived from embryonic stemcells. Embryonic stem cells in the present invention include embryonicstem cells derived from humans, monkeys, pigs, horses, cattle, sheep,dogs, cats, mice, rabbits or the like, and are preferably humanembryonic stem cells.

As used herein, the term “induced pluripotent stem cells” (iPSCs) refersto cells induced from differentiated cells by an artificialreprogramming process so as to have pluripotent differentiationpotential and is also referred to as reprogrammed stem cells. Theartificial reprogramming process may be performed by the use of avirus-mediated vector such as retrovirus or lentivirus or a nonviralvector or by introduction of nonvirus-mediated reprogramming factorsusing proteins and cell extractsor the like, or includes a reprogrammingprocess that is performed by stem cell extracts, compounds or the like.Induced pluripotent stem cells have properties almost similar to thoseof embryonic stem cells. Specifically, induced pluripotent stem cellsshow similarity in cell morphology and expression patterns of gene andprotein to those of embryonic stem cells, have pluripotency in vitro andin vivo, form teratomas, and generate chimeric mice upon injection intomouse blastocysts, and are capable of germline transmission of gene.Induced pluripotent stem cells in the present invention include thosederived from any animals, including humans, monkeys, pigs, horses, cows,sheep, dogs, cats, mice, rabbits, etc., and are preferably human inducedpluripotent stem cells.

As used herein, the term “reprogramming” or “dedifferentiation” refersto a process in which differentiated cells can be restored into cellshaving a new type of differentiation potential. In the presentinvention, the term “reprogramming” is used in the same meaning as cellreprogramming. This cell reprogramming mechanism involves the removal ofepigenetic (DNA state associated with changes in gene function thatoccur without a change in the nucleotide sequence) marks in the nucleus,followed by establishment of a different set of marks, and differentcells and tissues acquire different gene expression programs during thedifferentiation and growth of multicellular organisms.

As used herein, the term “promoting the reprogramming” means increasingthe rate of reprogramming or the efficiency of reprogramming in thereprogramming process. That is, the term includes increasing theefficiency of reprogramming in terms of speed and rate.

The inventive composition for promoting the reprogramming ofdifferentiated cells into pluripotent stem cells comprises RSC-133 at aconcentration that does not impair the survival and function of cells.Preferably, the composition comprises 0.01-50 μM of RSC-133. Morepreferably, it comprises 0.1-20 μM of RSC-133. Most preferably, itcomprises 8-12 μM of RSC-133.

The inventive composition for promoting the reprogramming ofdifferentiated cells into pluripotent stem cells may further comprisereprogramming factors. As used herein, the term “reprogramming factor”refers to a material that induces the reprogramming of differentiatedcells into induced pluripotent stem cells having a new type ofdifferentiation potential. The reprogramming factor may be any materialthat induces the reprogramming of differentiated stem cells, and it maybe selected depending on the kind of cells to differentiate. Preferably,the reprogramming factor that is used in the composition of the presentinvention may be one or more proteins selected from the group consistingof Oct4, Sox2, KlF4, c-Myc, Nanog and Lin-28 or one or more nucleic acidmolecules encoding these proteins. More preferably, the reprogrammingfactor may be Oct4 protein or a nucleotide molecule encoding theprotein. Particularly, the composition may comprise Oct4, Sox2 and KlF4proteins or nucleic acid molecules encoding these proteins.

The composition of the present invention is preferably in the form ofculture medium. Thus, materials that are generally contained in cellculture media may additionally be added to the composition of thepresent invention, as long as they do not interfere with thereprogramming of differentiated cells into pluripotent stem cells.

In an example of the present invention, the effects of theabove-described 39 trans-3-indoleacrylic acid-based compounds on thepromotion of induction of reprogramming were analyzed in mouse skinfibroblasts, and as a result, it was shown that the addition of thenovel compound RSC-133 or ID-558 effectively increased the efficiency ofreprogramming compared to a control (FIGS. 1A and 1B). When the effectsof the compounds were analyzed comparatively with knownreprogramming-promoting compounds (Valproic acid, 5-azacytidine,BIX-01294, etc.), it was shown that the novel compound RSC-133 mosteffectively increased the efficiency of reprogramming (FIGS. 2A and 2B).

In an example of the present invention, the effects of thirty-two noveltrasn-3-indoleacrylic acid-based compounds, including the novel compoundRSC-133 and ID-558 confirmed to effectively induce the reprogramming ofmouse skin fibroblasts, on the induction of reprogramming, wereanalyzed, and as a result, it was shown that the efficiency ofreprogramming of human skin fibroblasts treated with the novel compoundRSC-133 was effectively increased compared to that of a control group(FIG. 3).

In an example of the present invention, it was shown that the use of thenovel compound RSC-133 not only in normal culture conditions (21% O₂),but also in hypoxic culture conditions (5% O₂), increased the efficiencyof reprogramming compared to that of a control (FIGS. 4A and 4B).Several research groups reported that hypoxic culture conditions (5% O₂)are more effective in the maintenance of pluripotency and the inductionof reprogramming than normal culture conditions (21% O₂), and it wasfound that the addition of RSC-133 can additionally improve the cultureconditions of pluripotent stem cells and the conditions of reprogrammingof differentiated cells to pluripotent stem cells (FIGS. 4A and 4B).

In another example of the present invention, examination was carried outto determine whether the novel low-molecular-weight compound RSC-133 cansubstitute for an existing reprogramming factor (c-Myc) when thereprogramming of human skin fibroblasts into reprogrammed stem cells isinduced by addition of the compound RSC-133. It is known that c-Myc isan oncogenic gene and that the re-expression of c-Myc viral gene in thereprogrammed stem cells formed after induction of reprogramming isinvolved in carcinogenesis. Thus, studies on reprogramming methodsexcluding c-Myc have received attention. In the present invention, itwas found that, although RSC-133 alone does not show the effect ofsubstituting for c-Myc, the use of RSC-133 in combination with sodiumbutyrate (NaB) can substitute for c-Myc and can also increase theefficiency of reprogramming compared to that of a control (FIG. 5).

In conclusion, the novel low-molecular-weight compound RSC-133 wasconfirmed to be a factor that effectively increases the efficiency ofreprogramming of mouse and human somatic cells.

In still another example of the present invention, the efficiencies ofinduction of reprogramming at different concentrations of RSC-133 wereexamined to determine the optimal RSC-133 concentration range effectivefor increasing the efficiency of reprogramming, and as a result, it wasshown that the efficiency of reprogramming was increased in a mannerdependent on the concentration of RSC-133 (FIG. 8). Specifically,according to the culture conditions shown in the upper portion of FIG.8, human skin fibroblasts were transduced with OSKM virus, and then, asshown in the graph in the lower portion of FIG. 8, changes in theefficiency of reprogramming at different concentrations (0 μM, 0.1 μM, 1μM, 5 μM, 10 μM and 20 μM) of RSC-133 were examined. The efficiency ofreprogramming at each concentration of RSC-133 was determined bymeasuring the number of colonies that were positive inpluripotency-specific alkaline phosphatase (AP) staining and had ahESC-like morphology. The experimental results indicated that RSC-133could increase the efficiency of reprogramming in aconcentration-dependent manner at a concentration of up to 10 μM (FIG.8). It was shown that, when the concentration of RSC-133 was increasedto 20 μM, the efficiency of reprogramming was not additionally increased(FIG. 8). In addition, it was verified that, when human skin fibroblastswere treated with RSC-133 at a concentration up to 50 μM, RSC-133 didnot induce cytotoxicity (FIG. 9).

In still another example of the present invention, in order to optimizethe timing and period of RSC-133 treatment in a reprogramming protocol,RSC-133 was added under various conditions, and then changes in theefficiency of reprogramming were examined (FIG. 10).

As a result, it was shown that, when treatment with RSC-133 wasperformed for 5 days in each of four divided steps consisting of step 1(5 days after viral infection; condition 4 in FIG. 10), step 2 (5-10days after viral infection; condition 7 in FIG. 10), step 3 (10-15 daysafter viral infection; condition 8 in FIG. 10) and step 4 (15-20 daysafter viral infection; condition 9 in FIG. 10 9), treatment with RSC-133together with a reprogrammed-cell culture medium in step 1 (for 5 daysimmediately after viral infection) most effectively increased theefficiency of reprogramming (FIG. 10). In addition, it was shown that,when treatment with RSC-133 was performed for 10 days in each of threedivided steps consisting of step 1 (10 days after viral infection;condition 3 in FIG. 10), step 2 (5-15 days after viral infection;condition 6 in FIG. 10) and step 3 (10-20 days after viral infection;condition 10 in FIG. 10), treatment with RSC-133 in step 1 (for 10 daysimmediately after viral infection; culture in reprogrammed-cell culturemedium for 5 days, followed by culture in human embryonic stem cellculture medium in a fresh culture dish for days; condition 3 in FIG. 10)most effectively increased the efficiency of reprogramming (FIG. 10).Additionally, it was shown that, when treatment with RSC-133 wasperformed for 15 days in each of two divided steps consisting of step 1(15 days after viral infection; condition 2 in FIG. 10) and step 2 (5-20days after viral infection; condition 5 in FIG. 10), the efficiency ofreprogramming was increased in all the two conditions (FIG. 10). Amongall the conditions tested, continuous treatment with RSC-133 throughoutthe process of inducing reprogramming showed the highest increase in theefficiency of reprogramming (condition 1 in FIG. 10). In conclusion, itwas verified that, when treatment with RSC-133 is performed in theinitial stage of the reprogramming process, it showed the best effect onthe induction of reprogramming, and even treatment with RSC-133 isperformed after the initial stage of the reprogramming process, it cansignificantly increase the efficiency of reprogramming in a mannerdependent on the time of treatment.

In still another example of the present invention, whether the novelcompound RSC-133 has the effect of promoting the growth andproliferation of cells in a culture process for inducing reprogrammingwas examined by measuring the total number of cells at various timepoints (FIG. 11). It could be seen that an increase in the number ofcells transduced with OSKM was significantly slow compared to that of acontrol group not treated with OSKM and that treatment with the novelcompound RSC-133 under the same conditions promoted the growth of thecells (FIG. 12). In addition, a change in the proliferation of cellsduring a culture process for inducing reprogramming was examined by aBrdU assay. As a result, it was shown that treatment with the novelcompound RSC-133 significantly improved the cell growth in a mannerdependent on the time of treatment, similar to the above-describedresults (FIGS. 13 to 15).

In still another example of the present invention, in order to examinewhether the novel compound RSC-133 can promote kinetics in a cultureprocess for reprogramming to shorten the time required for the inductionof reprogramming, the expression patterns of pluripotency-specificmarkers were analyzed at various time points. As can be seen in FIGS. 15and 16, the results of immunostaining analysis (FIG. 15) and real-timePCR analysis (FIG. 16) indicated that, when reprogramming was inducedafter treatment with the novel compound RSC-133, the expression ofpluripotency-specific markers (Nanog, Tra1-81, Oct4, and Rex1) appearedearly than that in an untreated control group, suggesting that the novelcompound RSC-133 can stimulate reprogramming kinetics to effectivelyshorten the time required for the induction of reprogramming.

In still another example of the present invention, in order to examinewhether the novel compound RSC-133 can alleviate the senescence that isinduced by OSKM transduction known as an obstacle in the process forinducing reprogramming, changes in the expression patterns of p53, p21and p16 that are signaling factors known to play an important role inthe induction of aging were measured (FIG. 16) and a SA-β-Gal assay wasperformed (FIG. 17). As can be seen in FIG. 16, the aging senescencefactors p53, p21 and p16 were effectively inhibited in culture mediumcontaining the novel compound RSC-133, and as can be seen in FIG. 17,treatment with RSC-133 resulted in a significant decrease inSA-β-Gal-positive cells. These results suggest that the inhibition ofsenescence factors by RSC-133 contributes to improvement in conditionsof the induction of reprogramming.

In still another example of the present invention, in order to examinewhether the novel compound RSC-133 is associated with an epigeneticchange required for the reprogramming process, H3K9 acetylation that isepigenetic activation associated with the pluripotent state of stemcells was analyzed. As can be seen in FIG. 18, the results of Westernblot analysis by immunostaining indicated that H3K9 acetylation wasincreased in culture medium containing the novel compound RSC-133,suggesting that RSC-133 induces epigenetic activation in thereprogramming process. In addition, the results of measurement ofenzymatic activity and Western blot analysis indicated that the increasein H3K9 acetylation is associated with the inhibition of HDAC(particularly HDAC1) activity (FIG. 19) and HDAC1 expression (FIG. 21),which is induced by RSC-133. In addition, it was shown that the activityof DNA methyl transferase 1 (DNMT1) contributing to DNA methylation wassignificantly inhibited by RSC-133 (FIG. 20).

In still another example of the present invention, it was found thatpluripotent stem cells produced using medium containing the novelcompound RSC-133 maintained their hESC-like morphology during thecontinuous culture process and expressed pluripotency-specific markersat levels similar to those in hESC (FIGS. 22 and 23) and showed themethylation of pluripotency-specific promoters (Oct4 and Nanog) (FIG.24). In addition, it was verified that four transgenes (OSKMs) used inthe induction of reprogramming were all integrated into the genome ofthe host cells (FIG. 25) and that the pluripotent stem cells maintaineda normal karyotype (FIG. 26) and has the ability to differentiate intothree germ layers in vitro and in vivo (FIG. 27).

In another aspect, the present invention provides an in vitro cellculture comprising: differentiated cells; the above-describedcomposition for promoting reprogramming; and a reprogramming factor,wherein the differentiated cells are reprogrammed into pluripotent stemcells.

More specifically, the present invention encompasses all in vitro cellcultures obtained by treating differentiated cells with thereprogramming-promoting composition comprising the compound of formula 1and with a reprogramming factor. The in vitro cell culture alsocomprises various cells which are in the reprogramming process, variousproteins, enzymes and transcripts which are obtained during culture ofthe cells, and culture media containing them. Preferably, the compoundof formula 1 may be 3-[3-(1H-indol-3-yl)-acrylamido]-benzamide (RSC-133)or a pharmaceutically acceptable salt thereof.

The differentiated cells and pluripotent stem cells of the presentinvention are as described above.

The reprogrammed cells can show characteristics in that the function ofHDAC1 is inhibited and the level of H3K9ace is increased, compared to acontrol group not treated with the composition.

In still another aspect, the present invention provides a method forproducing reprogrammed stem cells from differentiated cells, the methodcomprising the steps of: (a) transferring a reprogramming factor to thedifferentiated cells; and (b) culturing the differentiated cells in amedium containing the above-described composition for promotingreprogramming.

Step (a) of transferring the reprogramming factor to the differentiatedcells may be performed by any method that is generally used in the artto provide nucleic acid molecules or proteins to cells. Preferably, step(a) may be performed by a method of adding the reprogramming factor to aculture of the differentiated cells, a method of injecting thereprogramming factor directly into the differentiated cells, or a methodof infecting the differentiated cells with a virus obtained frompackaging cells transfected with a viral vector including a gene of thereprogramming factor.

The method of injecting the reprogramming factor directly into thedifferentiated cells may be performed using any method known in the art.This method can be suitably selected from among microinjection,electroporation, particle bombardment, direct injection into muscles, aninsulator-based method, and a transposon-based method, but is notlimited thereto.

The reprogramming factor that is used in the present invention is asdescribed above.

In the present invention, the packaging cells may be selected from amongvarious cells known in the art depending on the kind of viral vectorused. Preferably, the packaging cells may be GP2-293 packaging cells,but are not limited thereto.

In addition, the viral vector that is used in the present invention maybe selected from among vectors derived from retroviruses, for example,HIV (human immunodeficiency virus), MLV (murine leukemia virus), ASLV(avian sarcoma/leukosis), SNV (spleen necrosis virus), RSV (rous sarcomavirus), MMTV (mouse mammary tumor virus) or the like, lentiviruses,adenovirus, adeno-associated virus, herpes simplex virus, etc, but isnot limited thereto. Preferably, the viral vector may be a retrovirusevector. More preferably, it may be the retroviruse vector pMXs.

Steps (a) and (b) may be performed simultaneously, sequentially or inthe reverse order. The above-described method may further comprise astep of isolating embryonic stem cell-like colonies from the cultureresulting from step (b).

In still another aspect, the present invention provides a compositionfor maintaining and culturing pluripotent stem cells in anundifferentiated state, the composition comprising the compound offormula 1 or a pharmaceutically acceptable salt thereof. In the presentinvention, pluripotent stem cells can be maintained in anundifferentiated state by the use of the composition comprising thecompound of formula 1 or a pharmaceutically acceptable salt thereof.

In an example of the present invention, H9 human embryonic stem cellsthat are typical human pluripotent stem cells were cultured inunconditioned medium (UM) in which the differentiation of the cellswould be easily induced, and the cells were treated with the novelcompound RSC-133 in order to examine whether the treatment of the cellswith the compound can inhibit the induction of differentiation of thecells and maintain the cells in an undifferentiated state. As can beseen in FIG. 28, in the culture medium treated with the novel compoundRSC-133, the induction of differentiation was inhibited, and thedifferentiated state was improved to the level of cells cultured inconditioned medium (CM). In addition, it was verified that, in theculture medium treated with RSC-133, the expression levels ofpluripotency-specific markers was maintained at the levels of thefactors in cells cultured in CM, and particularly, H3K9 acetylationassociated with epigenetic activation was maintained at a level similarto that of undifferentiated hESC cultured in CM (FIG. 29), suggestingthat RSC-133 is effective in maintaining and improving theundifferentiated state of human pluripotent stem cells.

In the present invention, the composition of formula 1 or apharmaceutically acceptable salt thereof may be used in an amountsufficient for maintaining pluripotent stem cells in an undifferentiatedstate. The cell culture medium for culturing undifferentiatedpluripotent stem cells preferably comprises RSC-133 in a concentrationof 0.01-50 μM. More preferably, it comprises RSC-133 at a concentrationof 0.1-20 μM. Most preferably, it comprises RSC-133 at a concentrationof 5-15 μM.

In addition, the cell culture medium according to the present inventionmay further comprise one or more selected from the group consisting ofan N2 supplement, a B27 supplement, bFGF and TGF. Herein, the N2supplement and the B27 supplement may be provided at a ratio of 1:1, andthe bFGF may be provided at a concentration of 4-100 ng/ml. Also, theTGF may be provided at a concentration of 1-10 ng/ml.

Moreover, the cell culture medium according to the present invention mayfurther comprise one or more components of a chemically defined mediumknown in the art. The addition of RSC-133 can improve the compositionand effect of the chemically defined medium.

In still another aspect, the present invention provides a method forestablishing an embryonic stem cell line capable of being maintained inan undifferentiated state, the method comprising the steps of: obtainingembryonic stem cells; and culturing the embryonic stem cells underculture conditions including the cell culture medium to obtain theembryonic stem cell line.

Herein, the embryonic stem cells include embryonic stem cells derivedfrom any animals, including humans, monkeys, pigs, horses, cattle,sheep, dogs, cats, mice, rabbits and the like. Preferably, the embryonicstem cells are human embryonic stem cells.

In still another aspect, the present invention provides a method ofculturing pluripotent stem cells in an undifferentiated state, themethod comprising culturing the pluripotent stem cells in a mediumcomprising the above-described cell culture medium for maintaining andculturing pluripotent stem cells in an undifferentiated state.

The use of the above-described method can maintain embryonic stem cellsin an undifferentiated state in the presence or absence of animal serumand feeder cells.

Herein, the embryonic stem cells include embryonic stem cells derivedfrom any animals, including humans, monkeys, pigs, horses, cattle,sheep, dogs, cats, mice, rabbits and the like. Preferably, the embryonicstem cells are human embryonic stem cells.

In still another aspect, the present invention provides an in vitro cellculture comprising: pluripotent stem cells; and the above-described cellculture medium serving to maintain the pluripotent stem cells in anundifferentiated state by a plurality of continuous subcultures.

The scope of the in vitro cell culture according to the presentinvention encompasses all in vitro cell cultures obtained by treatingpluripotent stem cells with the RSC-133-containing composition formaintaining and culturing pluripotent stem cells in an undifferentiatedstate. The in vitro cell culture also comprises various cells which arein a culture process, various proteins, enzymes and transcripts whichare obtained during culture of the cells, and culture media containingthem.

Pluripotent stem cells that are used in the present invention are asdescribed above.

Advantageous Effects

According to the present invention, when the novel low-molecular-weightcompound RSC-133 is added in a culture process for producingreprogrammed pluripotent stem cells from human differentiated cells, itcan increase the efficiency of reprogramming and can significantlyreduce the time required for the induction of reprogramming.Particularly, the novel compound RSC-133 can substitute for c-Myc actingas both a reprogramming factor and an oncogenic factor, and it caneffectively increase the efficiency of reprogramming in both normaloxygen culture conditions and hypoxic culture conditions. In addition,RSC-133 can inhibit the induction of aging occurring in thereprogramming process, exhibits the effect of promoting cellproliferation, and induces epigenetic activation to improve cultureconditions for induction of reprogramming. The present invention willcontribute to optimizing a process of producing induced pluripotent stemcells from a small amount of patient-specific somatic cells obtainedfrom various sources, and thus it will significantly improve a processof developing clinically applicable personalized stem cell therapyagents and new drugs and will facilitate the practical use of theseagents and drugs. In addition, the novel low-molecular-weight compoundRSC-133 can provide a cell culture medium effective for maintaining theundifferentiated state of human embryonic stem cells that are typicalpluripotent stem cells. The cell culture medium containing RSC-133 caneffectively induce the proliferation of human pluripotent stem cells inan undifferentiated state and can be effectively used for thedevelopment of a system for culturing large amounts of pluripotent stemcells.

DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B show a process of screening mouse cell reprogrammingfactors using analog library screening of novel trans-3-indoleacrylicacid-based compounds. In order to screen low-molecular-weight compoundsthat promote the production of reprogrammed stem cells, mouse embryonicstem cells (OG2-MEF; hemizygous for the Oct4-GFP transgene) weretransduced with reprogramming viruses (Oct4, Sox2, Klf4, and c-Myc).After 5 days, the cells were seeded on gelatin-coated plates and thencultured in mouse embryonic stem cell culture media containing each oflow-molecular-weight compounds until colonies having a morphologysimilar to that of mouse embryonic stem cells were formed. After 15days, the number of colonies expressing endogenous Oct4 GFP fluorescencewas measured, and compounds that increased the efficiency ofreprogramming compared to that of a control group were selected. FIG. 1Ashows an experimental process of inducing the reprogramming of mouseembryonic fibroblasts. Oct4 (O), Sox2 (S), Klf4 (K), and cMyc (M). FIG.1B is a graph showing the increase in efficiency of reprogramming byRSC-133 and ID-558 among screened compounds. The values shown on thegraph are mean values. The data are expressed as mean±SD (n=3).

FIG. 2a shows the results of comparatively analyzing the increase inreprogramming efficiency caused by the novel low-molecular-weightcompound 133. The reprogramming-promoting effects of a DNAmethyltransferase inhibitor (5-Azacytidine; AZA), a G9a histonemethyltransferase inhibitor (BIX-01294) and a histone deacetylaseinhibitor (Valproic acid; VPA), which are low-molecular-weight compoundsreported to promote the production of reprogrammed stem cells, and thenovel low-molecular-weight 133, were comparatively analyzed by measuringthe number of colonies expressing endogenous Oct4 GFP fluorescence.Treatment with RSC-133 greatly increased the efficiency of reprogrammingcompared to control group or treatment with other compounds orreprogramming of induction of reprogramming viruses (Oct4, Sox2, Klf4,and c-Myc (OSKM)). The values shown on the graph are mean values. Thedata are expressed as mean±SD (n=3) (**P<0.05).

FIG. 2b shows the results of comparatively analyzing the increase inreprogramming efficiency caused by the novel low-molecular-weightcompound 133. In a process of inducing the reprogramming of human skinfibroblasts by insertion of an OSKM reprogramming factor, the cells weretreated with AZA (0.5 μM), VPA (1 mM), TSA (20 nM), SB431542 (10 μM) andRSC 133 (10 μM), and the number of AP-positive colonies was counted tomeasure the efficiency of reprogramming. The values shown on the graphare mean values. The data are expressed as mean±SD (n=3).

FIG. 3 shows the results of screening reprogramming factors of humanskin fibroblasts (hFFs) using analog library screening of noveltrans-3-indoleacrylic acid-based compounds. In order to screenlow-molecular-weight compounds that promote the production ofreprogrammed stem cells, human fibroblasts were transduced withreprogramming viruses (Oct4, Sox2, Klf4, and c-Myc). After 5 days, thecells were seeded on Matrigel-coated plates, and then cultured inMEF-conditioned medium (CM) containing each of low-molecular-weightcompounds until colonies having a morphology similar to that of humanembryonic stem cells were formed. After 15 days, alkaline phosphatase(AP) activity was measured, and compounds that increased the efficiencyof reprogramming compared to that of a control group were selected.RSC-133 increased the efficiency of reprogramming of human skinfibroblasts, similar to that of mouse embryonic fibroblasts. The valuesshown on the graph are mean values. The data are expressed as mean±SD(n=3).

FIGS. 4A and 4B show that the novel low-molecular-weight compound 133increased the efficiency of reprogramming of human skin fibroblastscompared to that of a control group not only in normal conditions (21%O₂), but also in hypoxic conditions (5% O₂). According to previousreports, the efficiency of reprogramming increases in hypoxicconditions. The novel compound 133 can exhibit a synergistic effect inthe reprogramming process promoted by such hypoxic conditions. FIG. 4Ais a graph showing the results of measurement of AP activity. FIG. 4Bshows the morphology of reprogrammed stem cells induced by the additionof the novel compound 133 under hypoxic conditions.

FIG. 5 shows the results of examining whether the novellow-molecular-weight compound 133 shows the effect of substituting foran existing reprogramming factor (c-Myc) when the generation ofreprogrammed stem cells from human skin fibroblasts is induced byaddition of the novel low-molecular-weight compound 133. The compoundRSC-133 alone did not show the substituting effect, but addition of thecompound RSC-133 in combination with sodium butyrate (NaB) substitutedfor the c-Myc factor to increase the efficiency of reprogrammingcompared to that of a control group. The efficiency of reprogrammingobtained by measuring the number of AP-positive colonies is graphicallyshown. The values shown on the graph are mean values. The data areexpressed as mean±SD (n=3). The values shown on the graph are meanvalues.

FIG. 6 shows the ¹H NMR data of RSC-133.

FIG. 7 shows HPLC data relating to the purity of RSC-133.

FIG. 8 shows that RSC-133 increased the efficiency of reprogramming in amanner dependent on the concentration thereof in a culture medium forreprogramming. The upper panel schematically shows the experimentalprocess. The lower panel is a graph showing the efficiency ofreprogramming obtained by measuring the number AP-positive colonies as afunction of the concentration of RSC-133 treated. The values shown onthe graph are mean values. The data are expressed as mean±SD (n=3)(**P<0.005, by t-test).

FIG. 9 is a graph showing the results of analyzing the effect of RSC-133on the cytotoxicity of human skin fibroblasts. At 72 hours aftertreatment with various concentrations of RSC-133, cytotoxicity wasmeasured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (yellow tetrazole) assays. The values shown on the graph aremean values. The data are expressed as mean±SD (n=3).

FIG. 10 shows the results of examining a reprogramming process, in whichRSC-133 is involved, and an efficient reprogramming method utilizing thesame. The left panel schematically shows the experimental process. Theright panel shows the reprogramming efficiency measured by counting thenumber of AP-positive colonies after addition of RSC-133 duringdifferent periods of time in the reprogramming process. The values shownon the graph are mean values. The data are expressed as mean±SD (n=3)(*P<0.05, **P<0.005, by t-test).

FIG. 11 is a graph showing the effect of RSC-133 on cell growth during areprogramming process. The results were obtained in the same manner asthose in FIG. 10. The values shown on the graph are mean values. Thedata are are expressed as mean±SD (n=3).

FIG. 12 is a graph showing the results of examining the effect ofRSC-133 on the growth of human skin fibroblasts or human skinfibroblasts infected with a reprogramming factor (OSKM) virus. Thevalues shown on the graph are mean values. The data are mean±SD (n=3).

FIG. 13 shows that RSC-133 increased proliferating cell populationduring a reprogramming process. Immunocytochemical analysis wasperformed using monoclonal antibody for bromodeoxyuridine (BrdU), andthen percent BrdU positive cells are graphically shown. The values shownon the graph are mean values. The data are expressed as ±SD (n=3)(*P<0.05, by t-test).

FIG. 14 shows that RSC-133 increased proliferating cell population inhuman skin fibroblasts infected with reprogramming factor (OSKM) virus.The left panel is a set of graphs showing the results ofimmunocytochemical analysis performed using monoclonal antibody forBrdU, and the right panel is a graph showing the results of measurementof percent BrdU positive cells. The values shown on the graph are meanvalues. The data are expressed as mean±SD (n=3).

FIG. 15 is a set of photographs showing the results ofimmunocytochemical analysis performed using monoclonal antibodies forBrdU and undifferentiation markers (Nanog and Tra1-81) in cells culturedin the presence or absence of RSC-133. After induction of reprogramming,human skin fibroblasts were cultured for 10 days in the presence orabsence of RSC-133, followed by immunocytochemical analysis (scalebar=200 μm).

FIG. 16 shows the results of examining the effect of RSC-133 on areprogramming process. The relative expression levels of mRNA during areprogramming process were measured, and as a result, it was shown that,in a test group whose reprogramming was induced by treatment withRSC-133, the expressions of the pluripotency-specific markers Nanog,Oct4 and Rex1 were relatively increased, and the expressions of p53, p21and p16 known to inhibit reprogramming were relatively inhibited.

FIG. 17 shows the results of examining the effect of RSC-133 on cellcycle arrest (cell aging) in a reprogramming process. The degree of cellcycle arrest during a reprogramming process was measured by themeasurement of SA-β-Gal, and as a result, it was shown that cell cyclearrest was relatively reduced in a test group whose reprogramming wasinduced by treatment with RSC-133. The values shown on the graph aremean values. The data are expressed as mean±SD (scale bar=200 μm,**P<0.01).

FIG. 18 shows the effect of RSC-133 on histone acetylation (H3K9ace)during a reprogramming process. The left panel is a set of photographsshowing the results obtained by inducing the reprogramming of human skinfibroblasts, culturing the cells for 10 days in the presence or absenceof RSC-133, and then analyzing the cells by immunocytochemistry usingmonoclonal antibodies for H3K9ace and an undifferentiation marker(Nanog) (scale bar=200 μm). The right panel shows the results ofmeasuring the level of H3K9ace by Western blot analysis. H3 protein wasused as an internal control.

FIG. 19 shows the results of examining whether RSC-133 can inhibithistone deacetylase (HDAC) during the reprogramming of human skinfibroblasts. The left panel shows the results of measuring the activityof total HDAC enzymes during a reprogramming process performed in thepresence or absence of RSC-133. The right panel shows the amount ofHDAC1 protein measured after performing 10 days of culture in thepresence or absence of RSC-133. The values shown on the graph are meanvalues. The data are expressed as mean±SD (n=3) (*P<0.01, **P<0.005, byt-test).

FIG. 20 shows the results of examining the effect of RSC-133 on theactivity of DNA methyl transferase 1 (DNMT1) during the reprogramming ofhuman skin fibroblasts. The graph shows the activity of DNMT1 measuredafter culturing human skin fibroblasts transduced with OSKM, for 10 daysin the presence of RSC-133. For comparison, the activity of DNMT1 in theabsence of RSC-133 was set at 100%.

FIG. 21 shows the changes in amounts of HDAC-family proteins during thereprogramming of human skin fibroblasts in the presence or absence ofRSC-133. The upper panel schematically shows an experimental process.The lower panel shows the results of measuring the changes in amounts ofHDAC-family proteins by Western blot analysis. GAPDH protein was used asan internal control.

FIG. 22 shows the results of immunostaining performed to analyze theexpression of pluripotency-specific markers in the reprogrammed stemcells (RSC133-iPS) reprogrammed from human skin fibroblasts by additionof RSC-133.

FIG. 23 shows the results of RT-PCR analysis performed to analyze theexpressions of pluripotency-specific marker genes and reprogrammingfactors in reprogrammed stem cells (RSC133-iPS) induced from human skinfibroblasts by addition of RSC-133. Semi-quantitative RT-PCR wasperformed using transgene-specific PCR primers that can determine therelative expression between Total, Endo and retrovirus expression(Trans) genes.

FIG. 24 shows the results of analyzing the promoter methylation patternsof Oct4 and Nanog transcription factors in the reprogrammed stem cells(RSC133-iPS) induced from human skin fibroblasts by addition of RSC-133,H9 human embryonic stem cells (hESs) and human skin somatic cells (hFFs)induced from human skin fibroblasts by addition of RSC-133. Thehorizontal row of circles indicates an individual sequence from oneamplicon. The empty circles and the black circles indicate demethylatedand methylated CpG, respectively, and the percentage (%) of methylatedCpG is shown.

FIG. 25 shows the results of analyzing the integration of genes into thegenome of reprogrammed stem cells (RSC133-iPS) reprogrammed from humanskin fibroblasts by addition of RSC-133.

FIG. 26 shows that reprogrammed stem cells (RSC133-iPS) reprogrammedfrom human skin fibroblasts by addition of RSC-133 maintained a normalkaryotype (46, XY).

FIG. 27 is a set of photographs showing teratoma formation thatdemonstrates the in vivo differentiation potential of reprogrammed stemcells (RSC133-iPS) reprogrammed from human skin fibroblasts by additionof RSC-133.

FIG. 28 shows the results of examining the function of RSC-133 that isinvolved in the acquisition of pluripotency. It was observed that, whenH9 human embryonic stem cells (hESs) were cultured in unconditionedmedium (UM), the differentiation thereof was induced, but when the cellswere cultured in UM containing RSC-133, the degree of induceddifferentiation was inhibited, and pluripotency was acquired again. Theefficiency of acquisition of pluripotency was measured by calculatingthe number of colonies showing AP activity. The values shown on thegraph are mean values. The data are expressed as mean±SD (n=3) (*P<0.01,**P<0.005, by t-test).

FIG. 29 shows the results of Western blot analysis performed to examinethe change in amount of the pluripotency-specific markers Oct4 orH3K9ace in H9 human embryonic stem cells cultured in the presence orabsence of RSC-133. Human skin fibroblasts (hFFs) were used as anegative control group. H3 and GAPDH proteins were used as internalcontrols.

MODE FOR INVENTION

Hereinafter, the present invention will be described in further detailwith reference to examples. It is to be understood, however, that theseexamples are for illustrative purposes only and are not intended tolimit the scope of the present invention.

Example 1: Group of Low-Molecular-Weight Compounds

1-1. Method for Synthesis of Low-Molecular-Weight Compounds

Low-molecular-weight compounds, including RSC-133(3-(3-1H-indol-3-yl-acryloylamino)-benzamide), were synthesized by themethod shown in the following reaction scheme 1:

The reactants and reaction conditions in reaction scheme 1 are asfollows: a) 2a is H₂SO₄; 2b to 2e are i) SOCl₂, MeOH; ii) BnBr, K₂CO₃,DMF; iii) LiOH, THF, H₂O; iv) NH₄Cl, EDC, HOBt, DIPEA, DMF (2b); NH₂CH₃,NH₂CH(CH₃)₂, or NH(CH₃)₂, 50% PPAA, Et₃N, acetonitrile (2c-e); b) H₂, 5%Pd/C, MeOH; c) 5a to 5b are NH₂CH₃, or NH(CH₃)₂, 50% PPAA, Et₃N,acetonitrile; d) H₂, 10% Pd/C, MeOH; e) EDC, HOAt, or HOBt, DIPEA, DMF;PyBOP, DIPEA, DMF; DCC, DIPEA, THF; f) LiOH, THF/H₂O; g) 10a to 10c arefurfuryamine, 2-piperidin-1-yl-ethylamine, or3-morpholin-4-yl-propylamine, HATU, DIPEA, DMF; 10d is 2-bromopropane,ionic liquid, DMF.

In this synthesis process, all commercial compounds were grade-1reagents and were used without additional purification. Solutions weredried according to standard procedures. All reactions were performed ina flame-dried glass apparatus in an atmosphere of dry argon at apressure of 1 atm. Quantum nuclear magnetic resonance (1H-NMR) spectrawere measured in Varian (400 MHz or 300 MHz) spectrometer. Unlessotherwise specified, materials resulting from all reactions werepurified either by flash column chromatography using silica gel 60(230-400 mesh Kieselgel 60) or by thin layer chromatography usingglass-backed silica gel plates (1 mm thickness). In addition, reactionswere monitored by thin layer chromatography on 0.25 mm silica plates (E.Merck, silica gel 60 F254). Chromatograms were visualized by exposure toiodine vapor and immersion in PMA or Hanessian solution, followed byexposure to UV light. Isopropyl 4-hydroxy-3-nitrobenzoate correspondingto compound 2a in the above reaction scheme was prepared in thefollowing manner. First, sulfuric acid (98% H₂SO₄) was added dropwise toa solution of 4-hydroxy-3-nitrobenzoic acid (2.0 g, 10.9 mmol) in2-propylalcohol (25 mL) at 0° C., and the solution was refluxed in thepresence of argon for 48 hours. After completion of the reaction, thereaction mixture was concentrated under reduced pressure. The resultingmaterial was purified by silica gel column chromatography (n-hexane:EtOAc=5:1) to afford 2.11 g of isopropyl 4-hydroxy-3-nitrobenzoate (2a)as a yellow solid. ¹H NMR (CDCl₃, 300 MHz) δ=10.87 (s, 1H), 8.79 (d,J=1.2 Hz, 1H), 8.23 (dd, J=1.8 Hz, 8.7 Hz, 1H), 7.22 (t, J=11.7 Hz, 1H),5.30 (s, 2H), 5.26 (m, 1H), 1.38 (d, J=6.6 Hz, 6H).

4-Benzyloxy-3-nitrobenzamide corresponding to compound 2b in the abovereaction scheme was prepared in the following manner. First, to asolution of 4-benzyloxy-3-nitrobenzoic acid 1-3 (1 g, 3.6 mmol) andammonium chloride (294 mg, 5.5 mmol) in DMF (15 mL), EDC (842 mg, 4.4mmol), HOBt (593 mg, 4.4 mmol) and DIPEA (1.6 mL, 9.15 mmol) were added.The reaction solution was stirred overnight at room temperature andcooled with water, followed by extraction with EA. The resultingmaterial was purified by silica gel column chromatography (n-hexane:EA=7:3) to afford the desired compound (980 mg). ¹H NMR (CDCl₃, 400 MHz)δ=8.39 (d, J=2.4 Hz, 1H), 8.08 (dd, J=2.0 Hz, 8.4 Hz, 1H), 7.32-7.46 (m,5H), 7.16 (d, J=8.8 Hz, 1H), 5.30 (s, 2H).

Amide compounds 2c to 2e in the above reaction scheme were prepared inthe following manner. 4-benzyloxy-3-nitrobenzoic acid (500 mg, 1.0equiv) was suspended in acetonitrile and Et₃N (4.0 equiv), and 50% PPAA(1.2 equiv) was added thereto. The mixture was stirred at roomtemperature for 30 minutes, and each of suitable amines was addedthereto. Then, the reaction solutions were stirred overnight, and thendried by evaporation. The resulting materials were purified by columnchromatography to afford compounds 2c-2e.

1) 4-Benzyloxy-N-methyl-3-nitrobenzamide (2c) was obtained as a whitesolid. ¹H NMR (CDCl₃, 300 MHz) δ=8.24 (d, J=2.1 Hz, 1H), 7.99 (dd, J=2.4Hz, 9.0 Hz, 1H), 7.34-7.46 (m, 5H), 7.17 (d, J=8.7 Hz, 1H), 6.14 (br s,1H), 5.30 (s, 2H), 3.02 (d, J=5.1 Hz, 3H).

2) 4-Benzyloxy-N-isopropyl-3-nitrobenzamide (2d) was obtained as a whitesolid. ¹H NMR (CDCl₃, 300 MHz) δ=8.21 (d, J=2.1 Hz, 1H), 7.99 (dd, J=2.1Hz, 8.7 Hz, 1H), 7.32-7.46 (m, 5H), 7.16 (d, J=8.7 Hz, 1H), 5.89 (d,J=7.2 Hz, 1H), 5.30 (s, 2H), 4.27 (m, 1H), 1.27 (d, J=6.6 Hz, 6H).

3) 4-Benzyloxy-N,N-dimethyl-3-nitrobenzamide (2e) was obtained as awhite solid. ¹H NMR (CDCl₃, 300 MHz) δ=7.96 (d, J=1.5 Hz, 1H), 7.63 (dd,J=2.1 Hz, 8.7 Hz, 1H), 7.34-7.46 (m, 5H), 7.15 (d, J=8.7 Hz, 1H), 5.28(s, 2H), 3.07 (br s, 6H).

Compounds 2a to 2e were reduced in the following manner to preparecompounds 3a to 3e. Each of compounds 2a to 2e was dissolved inanhydrous MeOH, and then 5% palladium carbon was added thereto under anargon atmosphere. Each of the reaction solutions was stirred overnightin a hydrogen atmosphere and filtered through a celite pad, and thefiltrates were concentrated without additional purification to obtaincrude products.

1) Isopropyl 3-amino-4-hydroxybenzoate (3a) was obtained as a lightyellow solid from compound 2a. ¹H NMR (CDCl₃, 300 MHz) δ=7.43 (s, 1H),7.38 (d, J=8.7 Hz, 1H), 6.74 (d, J=8.1 Hz, 1H), 5.19 (m, 1H), 1.33 (d,J=6.6 Hz, 6H).

2) 3-Amino-4-hydroxybenzamide (3b) was obtained as a brown semi-solidfrom compound 2b. ¹H NMR (DMSO-d6, 400 MHz) δ=7.48 (s, 1H), 7.11 (d,J=8.7 Hz, 1H), 6.95 (dd, J=2.0 Hz, 8.4 Hz, 1H), 6.84 (br s, 1H), 6.61(d, J=8.4 Hz, 1H).

3) 3-Amino-4-hydroxy-N-methylbenzamide (3c) was obtained as a lightyellow solid from compound 2c. ¹H NMR (DMSO-d6, 300 MHz) δ=7.96 (d,J=4.5 Hz, 1H), 7.10 (d, J=2.1 Hz, 1H), 6.92 (dd, J=2.1 Hz, 8.1 Hz, 1H),6.63 (d, J=8.1 Hz, 1H), 4.61 (br s, 2H), 2.70 (d, J=4.5 Hz, 3H).

4) 3-Amino-4-hydroxy-N-isopropylbenzamide (3d) was obtained as a lightyellow solid from compound 2d. ¹H NMR (DMSO-d6, 300 MHz) δ=7.73 (d,J=8.1 Hz, 1H), 7.10 (d, J=1.5 Hz, 1H), 6.94 (dd, J=2.1 Hz, 8.1 Hz, 1H),6.62 (d, J=7.8 Hz, 1H), 4.60 (br s, 2H), 4.03 (m, 1H), 1.11 (d, J=6.6Hz, 6H).

5) 3-Amino-4-hydroxy-N,N-dimethylbenzamide (3e) was obtained as a lightyellow solid from compound 2e. ¹H NMR (DMSO-d6, 300 MHz) δ=9.36 (b, 1H),6.65 (d, J=1.5 Hz, 1H), 6.63 (d, J=8.4 Hz, 1H), 6.46 (dd, J=2.1 Hz, 7.8Hz, 1H), 4.64 (br s, 2H), 2.92 (s, 6H).

Amide compounds 5a to 5b in the above reaction scheme were prepared inthe following manner. 3-Nitrobenzoic acid (400 mg, 1.0 equiv) wassuspended in acetonitrile and Et₃N (4.0 equiv), and 50% PPAA (1.2 equiv)was added thereto. The mixture was stirred at room temperature for 30minutes, and each of suitable amines was added thereto. Then, thereaction solutions were stirred overnight, and then dried byevaporation. The resulting materials were purified by columnchromatography to afford compounds 5a-5b. 1)N-methyl-3-nitrobenzamide(5a) was obtained as a white solid.

¹H NMR (CDCl₃, 400 MHz) δ=8.58 (s, 1H), 8.35-8.37 (m, 1H), 8.16 (d,J=8.0 Hz, 1H), 5.19 (t, J=7.6 Hz 1H), 6.23 (br s, 1H), 3.07 (d, J=4.8Hz).

2) N,N-dimethyl-3-nitrobenzamide (5b) was obtained as a white solid. ¹HNMR (CDCl₃, 400 MHz) δ=8.29 (s, 1H), 8.27 (d, J=2.4 Hz, 1H), 7.77 (d,J=7.6 Hz, 1H), 7.62 (t, J=7.6 Hz 1H), 3.15 (s, 3H), 3.01 (s, 3H).

Meanwhile, methyl 3-aminobenzoate (6a) and 3-aminobenzamide (6b) arecommercially available.

Compound 5a or 5b was reduced in the following manner to preparecompound 6c or 6d. Nitrogen-substituted compound 5a or 5b was dissolvedin anhydrous MeOH, and 5% palladium carbon was added thereto under anargon atmosphere. The reaction solution was stirred overnight in ahydrogen atmosphere and filtered through a celite pad, and the filtratewas purified without additional purification to obtain a crude product.

1) 3-Amino-N-methylbenzamide (6c) was obtained as a white solid fromcompound 5a. ¹H NMR (DMSO-d6, 400 MHz) δ=8.13 (d, J=4.0 Hz, 1H),6.99-7.05 (m, 2H), 6.89 (d, J=7.2 Hz, 1H), 6.62-6.65 (m, 1H), 5.17 (s,2H), 2.71 (d, J=4.8 Hz, 3H).

2) 3-Amino-N,N-dimethylbenzamide (6d) was obtained as a white solid fromcompound 5b. ¹H NMR (DMSO-d6, 400 MHz) δ=7.02 (t, J=7.6 Hz, 1H),6.55-6.58 (m, 1H), 6.51 (s, 1H), 6.43 (d, J=7.6 Hz, 1H), 5.18 (s, 2H),2.89 (d, J=11.6 Hz, 6H).

The above-prepared compounds 3a to 3e or compounds 6a to 6d were reactedwith trans-3-indoleacrylic acid (7a) or 3-(1H-indole-3-yl)-propionicacid (7b) to obtain compounds 8a to 8j, compound 9 and compounds 10a to10d as shown in the above reaction scheme. Methods for preparing thesecompounds will be described in detail below.

1-2. Method for Synthesis of Low-Molecular-Weight Compound RSC-133 (8b)

Among the above-described low-molecular-weight compounds, RSC-133(3-[3-(1H-indol-3-yl)-acrylamido]-benzamide (8b)) was synthesized in thefollowing manner.

First, trans-3-indoleacrylic acid (7a) and 3-amino-benzamide (6b) weredissolved in DMF, andbenzotriazol-1-yl-N-oxy-tris(pyrrolidino)-phosphoniumhexafluorophosphate (PyBOP) and N,N-diisopropylethylamine (DIPEA) wereadded to the solution to perform a coupling reaction. The reactionsolution was stirred overnight at room temperature. The resultingmaterial was separated and purified to obtain3-[3-(1H-indol-3-yl)-acrylamido]-benzamido (RSC-133) as a yellow solid.The chemical characteristics and purity of RSC-133 were analyzed by ¹HNMR (FIG. 6) and HPLC (FIG. 7). ¹H NMR (CDCl₃, 300 MHz) d=8.90 (s, 1H),8.21 (s, 1H), 7.86-8.03 (m, 4H), 7.76 (d, J=8.1 Hz, 1H), 7.36-7.41 (m,3H), 7.20 (m, 2H), 6.60 (d, J=15.3 Hz, 2H), 3.88 (s, 3H).

RSC-133 (3-[3-(1H-indol-3-yl)-acrylamido]-benzamide) synthesized in thisExample has a structure of the following formula 2:

1-3. Synthesis of Low-Molecular-Weight Compound ID-52 (8a)

Among the above-described low-molecular-weight compounds, ID-52(3-(3-1H-indol-3-yl-acryloylamino)-benzoic acid methyl ester (8a)) wasprepared in the following manner. Trans-3-indoleacrylic acid (7a, 150mg, 0.8 mmol) and 3-amino-benzoic acid methyl ester (6a, 218 mg, 1.44mmol) were dissolved in DMF, and1-[3-(dimethyamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC, 230mg, 1.2 mmol), hydroxy-7-azabenotriazole (HOAT, 163 mg, 1.2 mmol) andN,N-diisopropylethylamine (DIPEA, 0.21 mL, 1.2 mmol) were added to thesolution to cause a coupling reaction. The reaction solution was stirredovernight at room temperature. Then, the resulting material wasseparated and purified to obtain3-(3-1H-indol-3-yl-acryloylamino)-benzoic acid methyl ester (ID-52) as ayellow solid. ¹H NMR (CDCl₃, 300 MHz) d=8.90 (s, 1H), 8.21 (s, 1H),7.86-8.03 (m, 4H), 7.76 (d, J=8.1 Hz, 1H), 7.36-7.41 (m, 3H), 7.20 (m,2H), 6.60 (d, J=15.3 Hz, 2H), 3.88 (s, 3H).

1-4. Synthesis of Low-Molecular-Weight Compound ID-1027 (8c)

Among the above-described low-molecular-weight compounds, ID-1027(3-[3-(1H-indol-3-yl)-acrylamido]-N-methylbenzamide (8c)) was preparedin the following manner. Trans-3-indoleacrylic acid (7a, 300 mg, 1.6mmol) and 3-amino-N-methylbenzamide (6c, 240 mg, 1.6 mmol) weredissolved in DMF, and PyBOP (1.7 g, 3.2 mmol) and DIPEA (0.84 mL, 4.8mmol) were added thereto. The reaction solution was stirred overnight atroom temperature and fractionated with EA and brine. The organic phasefraction was dried with MgSO₄ and concentrated. The resulting materialwas purified to obtain3-[3-(1H-indol-3-yl)-acrylamido]-N-methylbenzamide as a yellow solid. ¹HNMR (CD₃OD, 400 MHz) d=8.08 (s, 1H), 7.98 (d, J=15.2 Hz, 1H), 7.91 (d,J=8.0 Hz, 1H), 7.88 (dd, J=1.6 Hz, J=8.4 Hz, 1H), 7.69 (s, 1H),7.45-7.47 (m, 2H), 7.21-7.27 (m, 2H), 6.78 (d, J=15.6 Hz, 1H).

1-5. Synthesis of Low-Molecular-Weight Compound ID-1028 (8d)

Among the above-described low-molecular-weight compounds, ID-1028(3-[3-(1H-indol-3-yl)-acrylamido]-N,N-dimethylbenzamide (8d) wasprepared in the following manner. Trans-3-indoleacrylic acid (7a, 300mg, 1.6 mmol) and 3-amino-N,N-dimethylbenzamide (6d, 262.7 mg, 1.6 mmol)were dissolved in DMF, and PyBOP (1.7 g, 3.2 mmol) and DIPEA (0.84 mL,4.8 mmol) were added thereto. The reaction solution was stirredovernight at room temperature and fractionated with EA and brine. Theorganic phase fraction was dried with MgSO₄ and concentrated. Theresidue was purified to afford3-[3-(1H-indol-3-yl)-acrylamido]-N,N-dimethylbenzamide as a yellowsolid. ¹H NMR (CD₃OD, 400 MHz) d=7.96 (d, J=8.0 Hz, 1H), 7.92 (d, J=16.0Hz, 1H), 7.84 (s, 1H), 7.73 (d, J=7.6 Hz, 1H), 7.64 (s, 1H), 7.40-7.45(m, 2H), 7.18-7.25 (m, 2H), 7.13 (d, J=7.6 Hz, 1H), 6.78 (d, J=15.6 Hz,1H), 3.11 (s, 3H), 3.04 (s, 3H).

1-6. Synthesis of Low-Molecular-Weight Compound ID-134 (8e)

Among the above-described low-molecular-weight compounds, ID-134(3-[3-1H-indol-3-yl-propionylamino]benzamide (8e) was prepared in thefollowing manner. 3-(1H-indol-3-yl)-propionic acid (7b, 189 mg, 1.0mmol) and 3-amino-benzamide (6b, 68.1 mg, 0.5 mmol) were dissolved inDMF, and PyBOP and DIPEA were added to the solution to perform acoupling reaction. The reaction solution was stirred overnight at roomtemperature. Then, the resulting material was separated and purified toobtain 3-[3-1H-indol-3-yl-propionylamino]benzamide (ID-134) as a whitesolid. ¹H NMR (DMSO-d₆, 300 MHz) d=8.02 (s, 1H), 7.75 (d, J=7.8 Hz, 1H),7.50 (m, 2H), 7.32 (m, 2H), 6.93-7.09 (m, 3H), 3.04 (t, J=7.8 Hz, 2H),2.68 (t, J=7.2 Hz, 2H).

1-7. Synthesis of Low-Molecular-Weight Compound ID-514 (8f)

The low-molecular-weight compound ID-514 (isopropyl3-[3-(1H-indol-3-yl)-acrylamido]-4-hydroxybenzoate (8f)) was prepared inthe following manner. Trans-3-indoleacrylic acid (7a, 188 mg, 1.01 mmol)and isopropyl 3-amino-4-hydroxybenzoate (3a, 295 mg, 1.51 mmol) weredissolved in DMF, and EDC (290 mg, 1.51 mmol) and 1-hydroxybenzotriazolehydrate (HOBt, 204 mg, 1.51 mmol) were added thereto. The reactionsolution was stirred overnight at room temperature. The resultingmaterial was separated and purified to obtain isopropyl3-[3-(1H-indol-3-yl)-acrylamido]-4-hydroxybenzoate (ID-514) as a yellowsolid. ¹H NMR (DMSO-d₆, 300 MHz) d=11.65 (s, 1H), 11.07 (s, 1H), 9.60(s, 1H), 8.65 (d, J=1.5 Hz, 1H), 8.11 (d, J=6.6 Hz, 1H), 7.83 (d, J=2.1Hz, 1H), 7.79 (d, J=15.3 Hz, 1H), 7.57 (dd, J=1.5 Hz, 8.1 Hz, 1H), 7.47(d, J=7.5 Hz, 1H), 7.19-7.25 (m, 2H), 7.12 (d, J=15.3 Hz, 1H), 6.96 (d,J=8.1 Hz, 1H), 5.10 (m, 1H), 1.31 (d, J=6.6 Hz, 6H).

1-8. Synthesis of Low-Molecular-Weight Compound ID-1029 (8g)

The low-molecular-weight compound ID-1029(3-[3-(1H-indol-3-yl)-acrylamido]-4-hydroxybenzamide (8g)) was preparedin the following manner. Trans-3-indoleacrylic acid (7a, 300 mg, 1.6mmol) and 3-amino-4-hydroxybenzamide (3b, 243 mg, 1.6 mmol) weredissolved in DMF, and PyBOP (1.7 g, 3.2 mmol) and DIPEA (0.84 mL, 4.8mmol) were added thereto. The reaction solution was stirred overnight atroom temperature. The resulting material was separated and purified toobtain 3-[3-(1H-indol-3-yl)-acrylamido]-4-hydroxybenzamide (ID-1029) asa yellow solid. ¹H NMR (CD₃OD, 400 MHz) d=8.08 (s, 1H), 7.98 (d, J=15.2Hz, 1H), 7.91 (d, J=8.0 Hz, 1H), 7.88 (dd, J=1.6 Hz, J=8.4 Hz, 1H), 7.69(s, 1H), 7.45-7.47 (m, 2H), 7.21-7.27 (m, 2H), 6.78 (d, J=15.6 Hz, 1H).

1-9. Synthesis of Low-Molecular-Weight Compound ID-557 (8h)

The low-molecular-weight compound ID-557(3-[3-(1H-indol-3-yl)-acrylamido]-4-hydroxy-N-methylbenzamide (8h)) wasprepared in the following manner. DCC (220 mg, 1.07 mmol) and DIPEA (0.2mL, 1.07 mmol) were added to a solution of trans-3-indoleacrylic acid(7a, 100 mg, 0.53 mmol) in THF. The reaction solution was stirred atroom temperature for 30 minutes while3-amino-4-hydroxy-N-methylbenzamide (3c, 106 mg, 0.64 mmol) was addedthereto. The reaction solution was stirred overnight. The resultingmaterial was separated and purified to obtain3-[3-(1H-indol-3-yl)-acrylamido]-4-hydroxy-N-methylbenzamide (ID-557) asa yellow solid. ¹H NMR (acetone-d₆, 300 MHz) d=10.88 (b, 1H), 9.61 (b,1H), 8.03 (d, J=15.6 Hz, 1H), 8.02 (d, J=2.4 Hz, 1H), 7.94 (d, J=2.1 Hz,2H), 7.87 (d, J=2.4 Hz, 2H), 7.52-7.59 (m, 3H), 7.20-7.28 (m, 2H), 7.08(d, J=15.3 Hz, 1H), 6.93 (d, J=8.1 Hz, 1H), 3.62 (q, J=6.6 Hz, 1H), 2.88(d, J=4.5 Hz, 3H).

1-10. Synthesis of Low-Molecular-Weight Compound ID-556 (8i)

The low-molecular-weight compound ID-556(3-[3-(1H-indol-3-yl)-acrylamido]-4-hydroxy-N-isopropylbenzamide (8i))was prepared in the following manner. DCC (220 mg, 1.07 mmol) and DIPEA(0.2 mL, 1.07 mmol) were added to a solution of trans-3-indoleacrylicacid (7a, 100 mg, 0.53 mmol) in THF. The mixture was stirred at roomtemperature for 30 minutes while 3-amino-4-hydroxy-N-isopropylbenzamide(3d, 124 mg, 0.64 mmol) was added thereto. The reaction solution wasstirred overnight. The resulting material was separated and purified toobtain 3-[3-(1H-indol-3-yl)-acrylamido]-4-hydroxy-N-isopropylbenzamide(ID-556) as a yellow solid. ¹H NMR (acetone-d₆, 300 MHz) d=10.95 (b,1H), 10.89 (b, 1H), 9.61 (b, 1H), 8.02 (d, J=15.6 Hz, 1H), 8.01 (s, 1H),7.88 (t, J=3.0 Hz, 2H), 7.59 (dd, J=2.1 Hz, 8.7 Hz, 1H), 7.54 (dd, J=2.1Hz, 6.6 Hz, 1H), 7.20-7.28 (m, 3H), 7.08 (d, J=15.3 Hz, 1H), 6.91 (d,J=8.1 Hz, 1H), 4.21 (m, 1H), 1.22 (d, J=6.6 Hz, 6H).

1-11. Synthesis of Low-Molecular-Weight Compound ID-558 (8j)

The low-molecular-weight compound ID-558(3-[3-(1H-indol-3-yl)-acrylamido]-4-hydroxy-N,N-dimethylbenzamide (8j))was prepared in the following manner. DCC (220 mg, 1.07 mmol) and DIPEA(0.2 mL, 1.07 mmol) were added to a solution of trans-3-indoleacrylicacid (7a, 100 mg, 0.53 mmol) in THF. The mixture was stirred at roomtemperature for 30 minutes while 3-amino-4-hydroxy-N,N-dimethylbenzamide(3e, 116 mg, 0.64 mmol) was added thereto. The reaction solution wasstirred overnight. The resulting material was separated and purified toobtain 3-[3-(1H-indol-3-yl)-acrylamido]-4-hydroxy-N,N-dimethylbenzamide(ID-558) as a yellow solid. ¹H NMR (acetone-d₆, 300 MHz) d=10.85 (b,1H), 9.51 (b, 1H), 8.01 (d, J=15.3 Hz, 1H), 7.99-8.02 (m, 1H), 7.86 (d,J=2.1 Hz, 1H), 7.58 (d, J=2.1 Hz, 1H), 7.52-7.55 (m, 1H), 7.21-7.28 (m,2H), 7.17 (dd, J=1.8 Hz, 8.1 Hz, 1H), 7.03 (d, J=15.6 Hz, 1H), 6.93 (d,J=8.1 Hz, 1H), 3.02 (s, 6H).

1-12. Synthesis of Low-Molecular-Weight Compound ID-116(9)

The low-molecular-weight compound ID-116(3-[3-(1H-indol-3-yl)-acrylamido]benzoic acid) was prepared in thefollowing manner. 3-[3-(1H-indol-3-yl)-acrylamido]-benzoate (8a, 188 mg,0.59 mmol) was dissolved in THF/H₂O (1:1, 10 mL), and LiOH.H₂O (49.3 mg,1.17 mmol) was added thereto at room temperature. The reaction solutionwas stirred overnight at room temperature, after which it was acidifiedwith hydrochloric acid and extracted with ethyl acetate (EA). Theresulting material was separated and purified to obtain3-[3-(1H-indol-3-yl)-acrylamido]benzoic acid as a white solid. ¹H NMR(CD₃OD, 300 MHz) d=8.53 (s, 1H), 7.90-7.96 (m, 3H), 7.74 (d, J=7.8 Hz,1H), 7.58 (s, 1H), 7.41 (m, 2H), 7.19 (m, 2H), 6.78 (d, J=15 Hz, 1H).

1-13. Synthesis of Low-Molecular-Weight Compound ID-263 (10a)

The low-molecular-weight compound ID-263(3-[3-(1H-indol-3-yl)-acrylamido]-N-(furan-2-yl-methyl)benzamide) wasprepared in the following manner.3-(3-1H-indol-3-yl-acryloylamino)-benzoic acid (9, 500 mg, 1.63 mmol)and furfurylamine (0.23 mL, 2.45 mmol) were dissolved in DMF, and DIPEA(0.43 mL, 2.45 mmol) and HATU(O-(7-azabenzotriazol-1-yl)-N,N,N′N′-tetramethyluroniumhexafluorophosphate (931.6 mg, 2.45 mmol) were added thereto. Then, thereaction solution was stirred overnight at room temperature. Theresulting material was separated and purified to obtain3-[3-(1H-indol-3-yl)-acrylamido]-N-(furan-2-yl-methyl)benzamide (ID-263)as a yellow solid. ¹H NMR (CD₃OD, 300 MHz) d=8.13 (m, 1H), 7.96 (m, 2H),7.82 (m, 1H), 7.63 (s, 1H), 7.52 (m, 1H), 7.39-7.45 (m, 3H), 7.20 (m,2H), 6.78 (d, J=15.9 Hz, 1H), 6.32 (m, 2H), 4.56 (s, 2H).

1-14. Synthesis of Low-Molecular-Weight Compound ID-264 (10b)

The low-molecular-weight compound ID-264(3-(3-1H-indol-3-yl-acrylamino)-N-(2-piperidin-1-yl-ethyl)-benzamide)was prepared in the following manner.3-(3-1H-indol-3-yl-acryloylamino)-benzoic acid (9, 150 mg, 0.49 mmol)and 2-piperidin-1-yl-erthylamine (0.10 mL, 0.73 mmol) were dissolved inDMF, and HATU (277.6 mg, 0.73 mmol) and DIPEA (0.13 mL, 0.73 mmol) wereadded thereto. The reaction solution was stirred overnight at roomtemperature. The resulting material was separated and purified to obtain3-(3-1H-indol-3-yl-acryloylamino)-N-(2-piperidin-1-yl-ethyl)-benzamide(ID-264) as a yellow solid. ¹H NMR (DMSO-d₃, 300 MHz) d=11.67 (b, 1H),10.16 (s, 1H), 8.41 (s, 1H), 8.11 (s, 1H), 7.76-7.98 (m, 4H), 7.38-7.50(m, 3H), 7.23 (m, 2H), 6.84 (d, J=15.9 Hz, 1H) 3.33-3.43 (m, 6H), 2.51(m, 2H), 1.41-1.54 (m, 6H).

1-15. Synthesis of Low-Molecular-Weight Compound ID-265 (10c)

The low-molecular-weight compound ID-265(3-(3-1H-indol-3-yl-acrylamino)-N-(3-morpholin-4-yl-propyl)-benzamide)was prepared in the following manner.3-(3-1H-indol-3-yl-acryloylamino)-benzoic acid (9, 150 mg, 0.49 mmol)and 3-morpholin-4-yl-propylamine (0.11 mL, 0.73 mmol) were dissolved inDMF, and HATU (277.6 mg, 0.73 mmol) and DIPEA (0.13 mL, 0.73 mmol) wereadded thereto. The reaction solution was stirred overnight at roomtemperature. The resulting material was separated and purified to obtain3-(3-1H-indol-3-yl-acryloylamino)-N-(3-morpholin-4-yl-propyl)-benzamide(ID-265) as a yellow solid. ¹H NMR (DMSO-d₃, 300 MHz) d=11.66 (b, 1H),10.14 (s, 1H), 8.46 (ps-t, J=5.4 Hz, 1H), 8.09 (s, 1H), 7.76-7.98 (m,4H), 7.37-7.50 (m, 3H), 7.22 (m, 2H), 6.82 (d, J=15.9 Hz, 1H), 3.55-3.59(m, 4H), 3.27 (m, 1H), 3.16 (d, J=15.9 Hz, 1H), 2.31-2.36 (m, 6H) 1.69(m, 2H).

1-16. Synthesis of Low-Molecular-Weight Compound ID-517 (10d)

The low-molecular-weight compound ID-517 (isopropyl3-[3-(1H-indol-3-yl)acrylamido]benzoate) was prepared in the followingmanner. 3-(3-1H-indol-3-yl-acryloylamino)-benzoic acid (9, 180 mg, 0.59mmol) and ionic liquid (1.12 g, 1.47 mmol) were dissolved in DMF, and2-bromopropane (144 mg, 1.18 mmol) and DIPEA (152 mg, 1.18 mmol) wereadded thereto. The reaction solution was heated at 60° C. for 2 days andcooled with water. The resulting material was separated and purified toobtain isopropyl 3-[3-(1H-indol-3-yl)acrylamido]benzoate (ID-517) as alight yellow solid. ¹H NMR (DMSO-d₃, 300 MHz) d=11.68 (b, 1H), 10.22 (s,1H), 8.27 (s, 1H), 8.03 (d, J=9.0 Hz, 1H), 7.94-7.98 (m, 1H), 7.85 (s,1H), 7.80 (d, J=16.2 Hz, 1H), 7.62 (d, J=8.1 Hz, 1H), 7.47 (t, J=7.2 Hz,2H), 7.19-7.26 (m, 2H), 6.81 (d, J=15.6 Hz, 1H), 5.16 (m, 1H), 1.34 (d,J=6.0 Hz, 6H).

Example 2: Culture of Human Embryonic Stem Cells and Reprogrammed StemCells

Human embryonic stem cell (hESC) H9 (NIH Code, WA09; WiCell ResearchInstitute, Madison, Wis.) and reprogrammed stem cells (hiPSC) weregenerally cultured in hESC culture medium (composed of 80% DMEM/F12, 20%knockout serum replacement (KSR, Invitrogen, Carlsbad, Calif.), 1%non-essential amino acids (NEAA, Invitrogen), 1 mM L-glutamine(Invitrogen), 0.1 mM β-mercaptoethanol (Sigma, St. Louis, Mo.) and 6ng/ml bFGF (basic fibroblast growth factor, Invitrogen)) on γ-irradiatedMEFs (mouse embryonic fibroblasts). The cells were subcultured with 1mg/ml collagenase IV (Invitrogen) at intervals of 5-6 days. Humannewborn foreskin fibroblasts (hFF, ATCC, catalog number CRL-2097;American Type Culture Collection, Manassas, Va.) were cultured in DMEMcontaining 10% FBS (fetal bovine serum, Invitrogen), 1% non-essentialamino acid, 1 mM L-glutamine and 0.1 mM β-mercaptoethanol.

Example 3: Production of Retrovirus and Induction of hiPSC

A pMXs vector comprising the human cDNA of OCT4(POU5F1), SOX2,c-MYC(MYC) and KlF4, as disclosed in Takahashi, K. et al. (Cell 131,2007, 861-872), was purchased from Addgene. GP2-293 packaging cells weretransfected with retroviral vector DNA and a VSV-G envelop vector usingthe CalPhos transfection kit. At 24 hours after the transfection, thesupernatant containing the first virus was collected, and then themedium was replaced, and after 48 hours, the supernatant containing thesecond virus was collected.

For production of iPSC, human skin fibroblasts (hFFs) and mouseembryonic fibroblasts (MEFs) were seeded on gelatin-coated 6-well platesat a concentration of 1×10⁵ cells per well at one day beforetransfection and were transfected with virus in the presence ofpolybrene (8 μg/ml). At 5 days after the transfection, hFFs or MEFs werecollected by trypsin treatment and seeded again on Matrigel-coated6-well plates at a concentration of 5 to 6×10⁴ cells per well in orderto perform experiments in feeder-free conditions. The medium wasreplaced with MEF-CM medium containing 10 ng/ml of bFGF. MEF-CM wasprepared as γ-irradiated MEF according to a known method (Xu C. NatBiotechnol 19, 971-974), and 8 ng/ml bFGF was added to MEF-CM. Themedium was replaced at 2-day intervals. At 20 days after thetransfection, hESC-like colonies were collected and transferred to12-well plates having MEFs as feeder cells, and then continuouslycultured using the hESC culture method described in Example 2.

Example 4: Screening of Low-Molecular-Weight Compounds

The OSKM reprogramming factor virus was produced according to the methodof Example 3. In order to screen low-molecular-weight compounds thatincrease the efficiency of reprogramming, hFFs and mouse embryonicfibroblasts (OG2-MEF; hemizygous for the Oct4-GFP transgene) were seededon gelatin-coated 6-well plates at a concentration of 1×10⁵ cells at oneday before transfection, and then transfected with virus in the presenceof polybrene (8 μg/ml. At 5 days after the transfection, hFFs or MEFswere collected by trypsin treatment and seeded again on Matrigel- orgelatin-coated 12-well plates at a concentration of 3.5×10⁴ cells perwell in order to perform experiments under feeder-free conditions. Themedium was replaced with 10 ng/ml bFGF-containing MEF-CM or mouseembryonic stem cell culture medium, and then the preparedlow-molecular-weight compounds that are trans-3-indoleacrylic basedcompounds were added at various concentrations. The culture mediumcontaining each of the low-molecular-weight compounds was replaced at2-day intervals. At 15 days after the transfection, the number of eithercolonies showing AP activity or colonies expressing endogenous Oct4 GFPfluorescence was measured to determine the efficiency of reprogramming.

Example 5: RNA Extraction, Reverse Transcription and PCR Analysis

Total RNA was isolated from the produced cells using RNeasy Mini kit(Qiagen, Valencia, Calif.), and then reverse-transcribed usingSuperScript First-strand synthesis system kit (Invitrogen) according tothe manufacturer's instruction. Then, semi-quantitative RT-PCR wasperformed using platinum Tag SuperMix kit (Invitrogen) under thefollowing conditions: initial denaturation at 94° C. for 3 min, and then25-30 cycles, each consisting of 94° C. for 30 sec, 60° C. for 30 secand 72° C. for 30 sec, followed by final extension at 72° C. for 10 min.

Example 6: Alkaline Phosphatase (ALP) Staining

ALP staining was performed using a commercial ALP kit (Sigma) accordingto the manufacturer's instruction. Images of ALP-positive cells wererecorded using HP Scanjet G4010. In addition, bight field images wereobtained using an Olympus microscope (IX51, Olympus, Japan).

Example 7: Embryoid Body Differentiation

In order to measure the potential of hESC differentiation, humanembryoid bodies (hEBs) were cultured in hEB medium (DMEM/F12 containing10% serum replacement) in non-tissue culture treated Petri dishes. After5 days of growth in suspension, the embryoid bodies were transferred togelatin-coated plates and cultured in hEBs. The cells attached to thebottom of the plate were allowed to stand under the above-describedconditions for 15 days so as to differentiate while replacing themedium, if necessary.

Example 8: Immunocytochemistry

For immunostaining, cells were seeded on Matrigel-coated 4-well Lab-Tekchamber slides (Nunc, Naperville, Ill.) and cultured for 5 days underthe indicated conditions. The cells were fixed in 4% paraformaldehyde atroom temperature for 15 minutes, and then washed with PBS/0.2% BSA.Next, the cells were passed through PBS/0.2% BSA/0.1% Triton X-100 for15 minutes, and then incubated with 4% normal donkey serum (MolecularProbes, Eugene, Oreg., USA) in PBS/0.2% BSA at room temperature for 1hour. The cells were diluted with PBS/0.2% BSA, and then reacted withprimary antibody at 4° C. for 2 hours. After washing, the cells werereacted with FITC- or Alexa594-conjugated secondary antibody(Invitrogen) in PBS/0.2% BSA at room temperature for 1 hour. The cellswere counter-stained with 10 μg/ml DAPI. The chamber slide was observedwith an Olympus microscope or an Axiovert 200M microscope (Carl Zeiss,Gottingen, Germany).

Example 9: Analysis of Promoter Methylation of ReprogrammingTranscription Factors

In order to verify the characteristics of human embryonic stem cells andinduced pluripotent stem cells established using gene-transfectedretrovirus, promoter methylation of Oct3/4 and Nanog that are humanembryonic stem cell-specific transcription factors was analyzed. Toextract genomic DNA, reprogrammed stem cells and human embryonic stemcells, cultured in human embryonic stem cell media for 6 days, wereextracted using a DNA extraction kit (Qiagen Genomic DNA purificationkit). Bisulfite sequencing was performed in three steps. In the firststep, DNA was modified using sodium bisulfite, and in the second step,the gene region (generally promoter region) to be analyzed was amplifiedby PCR, and in the third step, the PCR product was sequenced todetermine the degree of methylation of DNA. The DNA modification processusing sodium bisulfite was performed using commercial EZ DNA MethylationKit (Zymo Research). When DNA is treated with bisulfite, methylatedcytosine does not change, whereas unmethylated cytosine is convertedinto uracil. Thus, when DNA is amplified by PCR using primers specificfor the nucleotide sequences of cytosine and uracil, methylated DNA andunmethylated DNA can be distinguished from each other. The primers usedare shown in Table 1 below.

TABLE 1 Gene Primer (Forward) Primer (Reverse) Accession No.For bisulfate sequencing bi Oct4-1 ATTTGTTTTTTGGGTAGTTAAAGGTCCAACTATCTTCATCTTAATA NM_002701 (SEQ ID NO: 1) ACATCC (SEQ ID NO: 2)bi Oct4-2 GGATGTTATTAAGATGAAGATAGTTGG CCTAAACTCCCCTTCAAAATC NM_002701(SEQ ID NO: 3) TATT (SEQ ID NO: 4) bi Nanog TGGTTAGGTTGGTTTTAAATTTTTGAACCCACCCTTATAAATTCTC NM_024865 (SEQ ID NO: 5) AATTA (SEQ ID NO: 6)

The PCR reaction mix consisted of 1 μg bisulfite-treated DNA, 0.25 mM/ldNTP, 1.5 mM/l MgCl2, 50 pM primer, 1×PCR buffer and 2.5U Platinum TaqDNA polymerase (Invitrogen, Carlsbad, Calif., USA) and had a finalvolume of 20 μl. The PCR reaction was performed under the followingconditions: initial denaturation at 95° C. for 10 min, and then 40cycles, each consisting of 95° C. for 1 min, 60° C. for 1 min and 72° C.for 1 min, followed by final extension at 72° C. for 10 min. The PCRreaction product was electrophoresed on 1.5% agarose gel, and after gelelectrophoresis, it was cloned into a pCR2.1-TOPO vector (Invitrogen).The nucleotide sequences of methylated and unmethylated DNAs wereanalyzed by sequencing using a M13 primer pair.

Example 10: Karyotype Analysis

Cultured human reprogrammed stem cells were analyzed by G-banding. Arepresentative image was obtained using ChIPS-Karyo (Chromosome ImageProcessing System, GenDix).

Example 11: Screening of Reprogramming Stimulating Compound (RSC) 133that is Novel Low-Molecular-Weight Compound that Increases theEfficiency of Reprogramming of Mouse and Human Cells

11-1. Discovery of Mouse Cell Reprogramming Factor by Analog LibraryScreening of Novel Trans-3-Indoleacrylic Acid-Based Compounds

To discover low-molecular-weight compounds that are involved in thereprogramming process, analog library screening of noveltrans-3-indoleacrylic acid-based compounds was performed in mouse cells.Mouse embryonic fibroblasts (OG2-MEF: hemizygous for the Oct4-GFPtransgene) were transfected with reprogramming viruses of Oct4, Sox2,Klf4 and c-Myc (OSKM), and after 5 days, the cells were seeded ongelatin-coated 12-well plates and incubated with mouse embryonic stemcell culture medium containing each of low-molecular-weight compoundsuntil colonies having a morphology similar to that mouse stem cells wereformed so that endogenous Oct4 GFP fluorescence was expressed (FIG. 1A).After 15 days, the number of colonies expressing endogenous Oct4 GFPfluorescence was measured to examine compounds that increased theefficiency of reprogramming compared to that of the control group, andas a result, it was shown that RSC-133 and ID-558 among the screenedcompounds showed the effect of increasing the efficiency ofreprogramming (FIG. 1B). Based on the results of the primary screening,novel indole-acrylic acid/indole-propionic acid derivatives (reactionscheme 1) were prepared, and structure-activity relationship (SAR) forthe reprogramming efficiency of the produced compounds was evaluated. Itwas found that, among the candidate compounds tested, indole-acrylicacid derivative compounds RSC-133 and ID-558 comprising an amino-freeindole ring coupled to a benzoic acid derivative by a double bond showedthe high reprogramming efficiency. In the benzoic acid derivativeportion of the two compounds, RSC-133 comprises simple benzamide, andID-558 comprises 4-hydroxy-N,N-dimethylbenzamide.

According to previous reports, a DNA methyltransferase inhibitor(5-Azacytidine), a G9a histone methyltransferase inhibitor (BIX-01294)and a histone deacetylase inhibitor (Valproic acid; VPA), which arelow-molecular-weight compounds, are known to promote the production ofreprogrammed stem cells. Interestingly, treatment with the novellow-molecular-weight compound RSC-133 greatly increased the efficiencyof reprogramming compared to treatment with the reported compounds orcompared to when increasing the MOI value of OSKM reprogramming virus(FIG. 2a ). The effect of RSC-133 on the promotion of reprogramming ofhuman cells was 1.5-3.3 times higher than those of AZA, VPA, TSA andSB431542 that are compounds known to promote the production of iPSC(FIG. 2b ).

11-2. Discovery of Reprogramming Regulators of Human Cells by AnalogLibrary Screening of Novel Trans-3-Indoleacrylic Acid-Based Compounds

To discover low-molecular-weight compounds that involved in thereprogramming of human somatic cells, reprogramming regulators of humanskin fibroblasts were investigated by analog library screening of thirtyselected novel trans-3-indoleacrylic acid-based compounds. Human skinfibroblasts were transfected with OSKM reprogramming virus, and after 5days, the cells were seeded on Matrigel-coated 12-well plates andincubated in MEF-conditioned medium (CM) containing each of thelow-molecular-weight compounds until colonies having a morphologysimilar to that of human embryonic stem cells were formed. After 15days, alkaline phosphatase (AP) activity was measured to determinecompounds that increased the efficiency of reprogramming compared tothat of the control group, and as a result, it was found that RSC-133increased the efficiency of reprogramming of human skin fibroblasts,similar to that of mouse embryonic fibroblasts (FIG. 3).

11-3. Examination of Effect of Novel Low-Molecular-Weight Compound 133on Increase in Efficiency of Reprogramming of Human Skin Fibroblasts

The novel low-molecular-weight compound 133 showed the effect ofincreasing the efficiency of reprogramming of human skin fibroblasts notonly in normal conditions (21% O₂), but also hypoxic conditions (5% O₂),compared to the control group (FIG. 4). The efficiency of reprogrammingincreases in hypoxic conditions (5% O₂), and the novel compound 133could exhibit a synergistic effect in the reprogramming processstimulated by such hypoxic conditions (5% O₂) (FIG. 4A) and could formreprogrammed stem cell colonies (FIG. 4B).

Meanwhile, examination was carried out to determine whether the novellow-molecular-weight compound 133 can substitute for an existingreprogramming factor (c-Myc) when the reprogramming of human skinfibroblasts is induced by the addition of the compound 133. It is knownthat c-Myc is an oncogene and the re-expression of c-Myc virus gene inreprogrammed stem cells formed after induction of reprogramming isinvolved in carcinogenesis. For this reason, studies on reprogrammingmethods excluding c-Myc have received attention. Interestingly, RSC-133alone did not show the substituting effect, but the addition of RSC-133in combination with sodium butyrate (NaB) substituted for c-Myc factorto increase the efficiency of reprogramming compared to that of thecontrol group (FIG. 5). In conclusion, it was found that the novellow-molecular-weight compound RSC-133 is a factor that increases theefficiency of reprogramming of mouse and human cells.

Example 12: Examination of Effect of Reprogramming Factor RSC-133 onConcentration-Dependent Induction of Reprogramming

Whether the effect of RSC-133 on the increase in the efficiency ofreprogramming is dependent on the amount of RSC-133 added was examined.Specifically, human skin fibroblasts were transfected with OSKM virus,and then, according to the experimental method shown in FIG. 8, whetherRSC-133 increases the efficiency of reprogramming in a manner dependenton the concentration thereof in a culture medium for reprogramming wasexamined. The efficiency of reprogramming was determined by measuringthe number of colonies showing AP activity. As a result, it could beseen that RSC-133 could increase the efficiency of reprogramming in aconcentration-dependent manner at a treatment concentration of up to 10μM (FIG. 8). The reason why the efficiency of reprogramming did notincrease at 20 μM is not that a high concentration of RSC-133 inducesthe cytotoxicity of human skin fibroblasts (FIG. 9). Thus, it can beseen that 10 μM of RSC-133 can increase the efficiency of reprogrammingof human skin fibroblasts to the highest level.

Example 13: Examination of Reprogramming Process in which RSC-133 isInvolved and Establishment of Efficient Reprogramming Using the Same

In order to examine the role of RSC-133 that is involved in thereprogramming process of human skin fibroblasts, RSC-133 was addedduring different periods of time in the reprogramming process as shownin the left panel of FIG. 10. The efficiency of reprogramming wasdetermined by counting the number of colonies showing AP activity. As aresult, when the cells were treated with RSC-133 for the same period oftime (5, 10 and 15 days) and when the cells were treated with RSC-133 inthe initial stage (including 5 days after viral infection) of thereprogramming process, the efficiency of reprogramming could besignificantly increased compared to those in other periods of time.However, when treatment with RSC-133 was continuously performedthroughout the reprogramming process, the efficiency of reprogrammingwas increased to the highest level (condition 1 in FIG. 10). Thus, itcan be seen that, although RSC-133 is involved in the initial stage ofreprogramming, continuous treatment with RSC-133 increases theefficiency of reprogramming to the highest level, suggesting thatRSC-133 is involved not only in the induction of reprogramming, but alsoin the maintenance of reprogrammed stem cells.

Example 14: Effect of RSC-133 on Promotion of Cell Growth

Recent reports indicated that the promotion of cell growth leads to anincrease in the efficiency of reprogramming. In addition, the growthrate of cells is associated with the self-renewal ability of embryonicstem cells. Thus, whether the novel compound RSC-133 can promote cellgrowth during reprogramming was examined. As a result, it could be seenthat when reprogramming was induced while the cells were treated withRSC-133, the growth rate of the cells increased by about 1,7 timescompared to that of the control group (FIG. 11). In addition, even whenreprogramming was not induced, RSC-133 showed a function of increasingthe growth rate of human skin fibroblasts (FIG. 12). In other words, inhuman skin fibroblasts or human skin fibroblasts which were transfectedwith reprogramming factor (OSKM) virus but the reprogramming of whichwas not induced, treatment with RSC-133 increased the growth of thecells (FIG. 12). Meanwhile, whether RSC-133 can increase proliferatingcell population was examined by immunocytochemical analysis using amonoclonal antibody for bromodeoxyuridine (BrdU). The results of theanalysis indicated that, during the reprogramming process (FIGS. 13 and16) and in human skin fibroblasts which were transfected withreprogramming factor (OSKM) virus but the reprogramming of which was notinduced (FIG. 14), treatment with RSC-133 increased BrdU-positive cellpopulation. This suggests that RSC-133 can promote cell growth toincrease the efficiency of reprogramming.

Example 15: Examination of Reprogramming Process Stimulated by RSC-133

In order to examine whether RSC-133 can increase the rate ofreprogramming, the relative levels of mRNA expressed during thereprogramming process were measured. Specifically, according to theexperimental method as shown in FIG. 10, human skin fibroblasts weretransfected with OSKM virus, and then the cells were harvested at 5-dayintervals, and the expression levels of genes in the cells cultured inthe presence or absence of RSC-133 were analyzed by real-time RT-PCR. Asa result, it was shown that, in the test group treated with RSC-133 toinduce reprogramming, the expression levels of the undifferentiationmarkers Nanog, Oct4 and Rex1 were relatively increased (FIG. 15 and theupper panel of FIG. 16), and the expression levels of p53, p21 and p16known to be involved in a reprogramming inhibitory mechanisms wererelatively inhibited (the lower panel of FIG. 16). In addition, at 10days after induction of reprogramming, the ratio of cellularsenescence-related β-galactosidase (SA-β-gal)-positive cells decreasedby 37.23% compared to that of an untreated control group (OSKM alone)(FIG. 17). Thus, it can be seen that RSC-133 can increase the efficiencyof reprogramming and also can be involved in the reprogramming processto stimulate kinetics.

Example 16: Analysis of Function of RSC-133 that is Involved in HistoneAcetylation During Reprogramming

Most of various low-molecular-weight compounds known to be involved inthe reprogramming process are involved genome methylation patterns,histone acetylation patterns or major signaling mechanisms. Among them,histone deacetylase (HDAC) inhibitors (VPA, TSA and SAHA) that areinvolved in the regulation of histone acetylation patterns are known toplay an important role in the reprogramming process. Particularly, anincrease in H3K9 acetylation (H3K9ace) levels is associated withpluripotency and reprogramming ability. As shown in FIG. 18,reprogramming of human skin fibroblasts was induced, and the cells werecultured for 10 days in the presence or absence of RSC-133, and thenanalyzed by immunocytochemistry using monoclonal antibodies for H3K9aceand an undifferentiation marker (Nanog), and as a result, the ratio ofundifferentiated cells having a high H3K9ace level was higher in thetest group treated with RSC-133 than in the control group (the leftpanel of FIG. 18). The same results were also obtained when the amountof protein was quantified by measuring the H3K9ace level by Western blotanalysis (the right panel of FIG. 18).

Next, whether the above-described results were results of the inhibitionof HDAC enzyme by RSC-133 during reprogramming of human skin fibroblastswas examined. As a result, it could be seen that the activity of totalHDAC enzymes was slightly inhibited by treatment with RSC-133 duringreprogramming (the left panel of FIG. 19). According to the experimentalmethod shown in FIG. 21, the expression levels of HDAC-family proteinswere measured, and as a result, it could be seen that the expressionlevel of HDAC1 in the test group treated with RSC-133 was inhibited at10 days after treatment with RSC-133 compared to that of the controlgroup (the light panel of FIG. 19 and FIG. 21). It was reported thatHDAC1 can occupy the Oct4, Sox2, Klf4 and c-Myc gene loci in embryonicstem cells and that the expression of pluripotency-specific marker genesin HDAC1-deleted embryonic stem cells is increased. Thus, it is believedthat RSC-133 can inhibit the function of HDAC1 and increase the H3K9acelevel to stimulate the reprogramming process.

Example 17: Analysis of Function of RSC-133 that is Involved in DNAMethylation During Reprogramming

RSC133 regulates the activities of epigenetic regulators such as DNAmethyltransferase (DNMT) and histone deacetylase (HDAC). At 10 daysafter induction of reprogramming by RSC133, the activity of DNMT1 wasreduced by about 38% compared to that in an untreated control group(OSKM alone) (FIG. 20). Epigenetic chromatin remolding by DNAmethylation and/or histone modification plays an important in a widerange of transcriptional regulation in the reprogramming process. WhenDNA methylation is inhibited by treatment with the DNA methylationinhibitor AZA or deletion of DNMT1, the conversion of the cells to iPSCis rapidly induced. The increased level of H3K9 histone acetylation(ace) is particularly associated with the restoration of pluripotencyand reprogramming ability. It is known that DNA methylation by DNMT1 hasa close connection with the change in chromatin status caused by HDACactivity. Putting these results together, it can be seen that theinhibition of DNMT1 and HDAC1 activity by RSC-133 changes DNAmethylation and chromatin status and increases accessibility to the lociof above-defined four reprogramming enzymes to have an indirect effecton the transcriptional regulation of the genes, thereby promoting thereprogramming process.

Example 18: Analysis of Characteristics of Reprogrammed Human Stem CellsInduced by RSC-133

18-1. Analysis of Expression of Pluripotency-Specific Markers

The stem cell characteristics of the reprogrammed stem cell lines(RSC133-iPS) induced from human skin fibroblasts by the addition ofRSC-133 were analyzed by ALP staining and immunostaining. Two cell lines(RSC133-iPS1 and RSC133-iPS2) were analyzed. As a result, the RSC133-iPScell lines were very similar such that they were not distinguishedmorphologically or by the ALP staining and immunostaining of humanembryonic stem cell markers (OCT4, NANOG, SSEA-3, SSEA-4, TRA-1-60, andTRA-1-81) (FIG. 22). In addition, the expressions of Oct4, Sox2, cMycand Klf4 mRNA were analyzed by semi-quantitative RT-PCR, and as aresult, it was shown that the RSC133-iPSs expressed Oct4, Sox2, cMyc andKlf4 at the total and endogenous levels similar to those in humanembryonic stem cells and that the silencing of the genes introduced byretroviruses was completely completed (FIG. 23).

18-2. Analysis of Methylation in Reprogrammed Stem Cells Induced byRSC-133

According to the method shown in Example 8, the degrees of the promotermethylation regions of the stem cell markers Oct4 and Nanog genes of theRSC133-iPSs were analyzed by bisulfite sequencing. As a result, as canbe seen in FIG. 21, the RSC133-iPSs showed demethylation patternssimilar to those of the human embryonic stem cells (H9), but the parentcells (hFFs) still maintained methylation (FIG. 24).

18-3. Analysis of Genomic Integration of Reprogrammed Stem Cells Inducedby RSC-133

Genomic integration of each of RSC133-iPS1 and RSC133-iPS2 was analyzed.Specifically, genomic DNA was extracted from each of the cell linesusing a DNeasy kit (Qiagen, Valencia, Calif.), and 300 ng of each of thegenomic DNAs was amplified by PCR using primers capable of specificallyamplifying the genomic DNA and the transferred gene. As a result, it wasfound that, in the RSC133-iPS cell lines, Oct4, Sox2, cMyc and Klf4 wereintegrated (FIG. 25). Herein, the human embryonic stem cell line (H9,hES) and the human skin fibroblast cell line (CRL2097, hFF) were used ascontrol groups.

18-4. Analysis of Karyotype of Reprogrammed Stem Cells Induced byRSC-133

The karyotype of the RSC133-iPSCs was analyzed by G-banding.Representative images were obtained using ChIPS-Karyo (Chromosome ImageProcessing System, GenDix) (FIG. 26). As a result, the reprogrammedRSC133-iPSCs showed a normal karyotype showed a normal karyotype (46,XY).

18-5. Examination of Pluripotent Ability of Reprogrammed Stem CellsInduced by RSC-133

In order to examine whether the reprogrammed stem cells (RSC133-iPS)from human fibroblasts by the addition of RSC-133 possessdifferentiation potential that is the feather of stem cells, thedifferentiation potential of embryoid bodies derived from each of thereprogrammed stem cell lines was examined. Specifically, the cells werecultured in suspension, and then the embryoid bodies were incubatedagain on gelatin-coated plates for 10 days under differentiationconditions, after which the expression of marker proteins that areexpressed specifically in cells that differentiated into three germlayers was analyzed by immunochemical staining. As a result, cellspositive to Tuj1 (exoderm), Nestin(exoderm), desmin (mesoderm), α-SMA(α-smooth muscle actin, mesoderm), Sox17 (endoderm) and FoxA2 (endoderm)were detected. Such results that the reprogrammed stem cells(RSC133-iPS) induced by RSC-133 had the capability to differentiate intothree germ layers and maintained pluripotency.

In addition, in order to examine the in vivo pluripotency of the humanreprogrammed stem cells (RSC133-iPS) induced by the addition of RSC-133,the reprogrammed stem cells RSC133-iPS were injected subcutaneously intothe dorsal flanks of immunodeficiency (SCID) mice. After about 12 weeks,teratomas could be observed, and neural rosette (exoderm),adipocyte(mesoderm), cartilage (mesoderm) and gut-like epithelium(endoderm) were observed in the teratoma by hematoxylin/eosin staining(FIG. 27). This suggests that the human reprogrammed stem cells(RSC133-iPSCs) induced by the addition of RSC-133 have the capability todifferentiate into three germ layers in vitro and in vivo.

Example 19. Effects of RSC-133 on Maintenance and Stimulation ofPluripotency

When human embryonic stem cells (H9) are cultured in unconditionedmedium (UM), their differentiation is induced. However, when the cellswere cultured for 6 days in UM supplemented with RSC-133, it could beobserved that the degree of induced differentiation was inhibited andpluripotency was re-acquired (the upper panel of FIG. 28). As a positivecontrol group, embryonic stem cells cultured for 6 days inMEF-conditioned medium (CM) effective for maintenance of anundifferentiated state were used. The efficiency of maintenance of anundifferentiated state and inhibition of differentiation was determinedby measuring the number of colonies showing AP activity (the lower panelof FIG. 28). In addition, the results of immunostaining at the proteinlevel indicated that, when human embryonic stem cells were cultured inUM supplemented with RSC-133, the pluripotency-specific markers (Oct4and Tra1-81) of the human embryonic stem cells were expressed at levelssimilar to those of human embryonic stem cells cultured in CM. Theinhibition of differentiation and maintenance of undifferentiation byRSC-133 was additionally verified by analysis of expression of thepluripotency-specific markers Oct4 and H3K9ace, and it was observed thatthe human embryonic stem cells cultured in UM containing RSC-133expressed Oct4 and H3K9ace at levels similar to those in the humanembryonic stem cells cultured in CM (FIG. 31). Thus, it was verifiedthat RSC-133 has effects not only on the reprogramming of human somaticcells, but also on the maintenance of pluripotency of human pluripotentstem cells.

Although the preferred embodiments of the present invention have beendescribed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

The invention claimed is:
 1. A method of culturing pluripotent stemcells in an undifferentiated state, comprising a step of culturing thepluripotent stem cells in a medium comprising a compound of thefollowing formula 1 or a pharmaceutically acceptable salt thereof:

wherein (i) R₁ is an amino group (NH₂) and R₂ is hydrogen (H) or (ii) R₁is a dimethylamino group (N(CH₃)₂) and R₂ is a hydroxyl group (OH; and aportion indicated by - - - is a double bond.
 2. The method of claim 1,wherein R₁ is an amino group (NH₂); R₂ is hydrogen; and the portionindicated by - - - is a double bond.
 3. The method of claim 1, whereinR₁ is a dimethylamino group (N(CH₃)₂); R₂ is a hydroxyl group (OH); andthe portion indicated by - - - is a double bond.
 4. The method of claim1, wherein the compound of formula 1 is selected from the groupconsisting of the following compounds: 1)3-[3-(1H-indol-3-yl)acrylamido]benzamide; and 2)3-[3-(1H-indol-3-yl)acrylamido]-4-hydroxy-N,N-dimethylbenzamide.
 5. Themethod of claim 1, wherein the pluripotent stem cells are embryonic stemcells or induced pluripotent stem cells.
 6. The method of claim 1,wherein the pluripotent stem cells are of human origin.
 7. The method ofclaim 1, wherein the concentration of the compound of formula 1 or apharmaceutically acceptable salt thereof is 0.01-20 μM.
 8. The method ofclaim 1, culturing pluripotent stem cells in an undifferentiated statecorresponds to an increase in Oct4 and H3K9ace level compared to thosein a control group not cultured in the composition.